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.

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.

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.

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.

A negative component superimposed on event-related potentials during light drowsiness.

The present study examined the nature of the negative shift of event-related potential (ERP) recorded during the fully awake state, wakefulness with minor awareness deficit (light drowsiness) and stage 1 of NREM sleep. The cortical responses evoked by two types of auditory stimuli were recorded in nine subjects at the different levels of consciousness described above. A negative component with peak latency of 250-350 msec, N300, was identified in ERP during light drowsiness but not in the fully awake state. In stage 1a (stage 1 without vertex sharp waves), the amplitude of N300 was higher than that in light drowsiness, and it was higher in stage 1b than in stage 1a. The scalp distribution of N300 was predominantly on the vertex. It also confirmed that the vertex sharp wave evoked during stage 1 is maximal on the vertex and its peak latency is approximately 300 msec. Considering the similarity in scalp distribution and peak latency between N300 and vertex sharp wave, it is possible that these electroencephalogram phenomena are generated by an identical synchronizing mechanism in the brain. We assumed that N300 observed during light drowsiness may be an incomplete product of vertex sharp wave.

[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.

The utility of three-dimensional computed tomography in unanticipated difficult endotracheal intubation.

IMPLICATIONS: We experienced a case of unanticipated difficult intubation with direct laryngoscopy because of narrowing of the retropharyngeal air space and laryngeal vestibulum. It is suggested that three-dimensional computed tomography is useful for evaluating both the abnormality of an airway and its relationship to surrounding tissue.

A negative component on event related potential recorded in the drowsy state.

Behavior of event related potential (ERP) components in the drowsy state was examined in nine subjects using oddball paradigm. A component with peak latency of 300 msec, N300, was superimposed on ERP in the drowsy state. N300 appeared also in stage 1 of NREM sleep and closely resembled vertex sharp wave evoked by sound stimulation in both scalp distribution and peak latency. It was suggested that N300 recorded in the drowsy state and vertex sharp wave recorded in stage 1 of NREM sleep are generated by the identical synchronizing mechanism in the brain.

Some sensory stimuli generate spontaneous K-complexes.

The present study was performed in order to determine whether spontaneous K-complex are induced by sensory stimuli. Electroencephalogram (EEG) segments in stage 2 sleep containing an evoked K-complex or spontaneous K-complex were separately averaged with respect to the peak of N300, one of the main components constituting the K-complex. Small negative and positive components were found immediately before the main components of spontaneous K-complex in averaged EEG. These two components were judged to correspond to N100 and P200 induced by the sound stimulus. The present findings suggest that the spontaneous K-complex is not a spontaneous phenomenon but that it is induced by sensory stimuli.

Cortical activity of REM sleep often occurs earlier than other physiological phenomena.

N300 appearing in response to sound stimulus was used as an index to determine the occurrence of cortical activity characterizing REM sleep. In 5/10 subjects, marked reduction of N300 amplitude occurred even in the period of 0.5-2.5 min immediately preceding the appearance of muscle atonia characterizing REM sleep. Neither muscle atonia nor rapid eye movements appeared prior to the marked reduction of N300 amplitude in any subject. This suggests that the cortical activity characterizing REM sleep sometimes occurs a few minutes (or less) earlier than other physiological phenomena.

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.

Electrophysiological evidence suggesting that sensory stimuli of unknown origin induce spontaneous K-complexes.

The present study was performed to determine whether or not spontaneous K-complexes are induced by sensory stimuli. In the first part of the present study, sound stimuli were prescribed during sleep in 7 healthy, young, adult subjects. EEG segments in stage 2 sleep were averaged separately according to the presence or absence of an evoked K-complex appearing after each stimulus. The sound stimulus induced N100 and P200 components in averaged EEGs regardless of K-complex appearance. The appearance of N100 and P200 components was considered to be an indicator of the presence of sensory stimuli. In the second part of the present study, EEG segments in stage 2 sleep containing an evoked K-complex or spontaneous K-complex were separately averaged with respect to the peak of N300, one of the main components constituting the K-complex. Small negative and positive components were found just before the main components of spontaneous K-complexes in averaged EEGs. These two components were judged to correspond to N100 and P200 components induced by the sound stimulus, as they appeared just before the main components of the spontaneous K-complex with almost the same lag time between the two components, or between each of the two components and the main components of K-complex, as in the case of N100 and P200 appearing just before the evoked K-complex. The present findings suggest that the spontaneous K-complex is not a spontaneous phenomenon, but that it is induced by sensory stimuli, probably of extracerebral origin.

K-complex evoked in NREM sleep is accompanied by a slow negative potential related to cognitive process.

Evoked cortical responses to two kinds of auditory stimuli (rare and frequent) were analyzed to determine whether or not a K-complex evoked in stage 2 of NREM sleep is accompanied by some endogenous cognitive components of the event-related potential. All the 7 subjects examined in this sleep state failed to provide the correct behavioral response to auditory stimuli, but a K-complex was evoked more frequently by rare stimuli than by frequent stimuli. EEG segments in stage 2 were averaged separately according to the presence or absence of K-complexes emerging just after the stimulation. In cases where K-complexes did not emerge, a long-lasting negative potential of relatively low voltage appeared in the difference wave, which was obtained by subtracting the averaged EEG for frequent stimuli from that for rare stimuli. In cases where K-complexes emerged, a similar long-lasting negative potential of large amplitude appeared in the difference wave. These data may indicate that a K-complex evoked by an external stimulus is accompanied by a potential related to a cognitive process, which appears with greater amplitude in cases where a K-complex is evoked.

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.

Cortical reactivity in REM sleep with tonic mentalis EMG activity induced by clomipramine: an evaluation by slow vertex response.

Slow vertex response (SVR) to sound stimuli was used as an index to assess whether cortical reactivity in REM sleep with tonic mentalis EMG activity (stage 1-REM) corresponds to that in REM sleep or rather to that in stage 1 of NREM sleep. In 11 young adult subjects 3 night polygraphic records were made after administration of 25 or 50 mg of clomipramine or non-active placebo. Stage 1-REM was observed in the drug night in 8 of the 11 subjects. N1-P2 and P2-N2 amplitudes of SVR were markedly smaller in stage 1-REM than in stage 1, but were virtually of the same height in stage 1-REM and in stage REM. This indicates that cortical reactivity in stage 1-REM closely resembles the counterpart in stage REM. This finding corresponds well with the frequent emergence of vivid dreams in stage 1-REM, indicating that stage 1-REM observed in the present subjects probably represents REM sleep without muscle atonia.

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.

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.