Endothelin receptor-mediated responses in trigeminal ganglion neurons.

Recent evidence implicates endothelin in nociception, but it is unclear how endothelin activates trigeminal ganglion (TRG) neurons. In the present study, we investigated the expression of the endothelin receptors ETA and ETB and endothelin-induced responses in rat TRG neurons. Double-immunofluorescence studies demonstrated that ETA and ETB were expressed in TRG neurons and that 26% of ETA- or ETB-expressing neurons expressed both receptors. During whole-cell patch-clamp recording, endothelin-1 enhanced an induced current in response to capsaicin, a TRPV1 agonist, in approximately 20% of dissociated neurons. The enhancement was blocked by the PKC inhibitor chelerythrine and by the ETA antagonist BQ-123, but not by the ETB antagonist BQ-788. Ca(2+)-imaging showed that endothelin-1 increased the intracellular Ca(2+) concentration in more than 20% of the dissociated neurons. Importantly, unlike the effect of endothelin-1 on capsaicin-induced current, the Ca(2+) response was largely suppressed by BQ-788 but not by BQ-123. These results suggest that ETA-mediated TRPV1 hyperactivation via PKC activation and ETB-mediated Ca(2+) mobilization occurs in different subsets of TRG neurons. These endothelin-induced responses may contribute to the induction of orofacial pain. The ETB-mediated function in TRG neurons is a special feature in the trigeminal system because of no ETB expression in dorsal root ganglion neurons.

The cellular and molecular mechanisms of bone invasion by oral squamous cell carcinoma.

Oral squamous cell carcinomas (SCCs) are malignant tumours that frequently invade the mandibular bone and bone invasion is a common clinical problem. Recent studies have revealed that bone resorption by osteoclasts is an important step in the process of bone invasion by oral SCCs. However, the cellular and molecular mechanisms of bone invasion by oral SCCs remain unclear. Oral SCCs invade the mandibular bone through an erosive, mixed or infiltrative pattern that correlates with clinical behaviours. The expressions of interleukin (IL)-6, IL-11, tumour necrosis factor (TNF) α and parathyroid hormone-related protein (PTHrP) were higher in the infiltrative pattern than in the erosive pattern. These cytokines lead to receptor activator of NF-κB ligand (RANKL) expression or osteoprotegerin (OPG) suppression not only in oral SCC cells but also in cancer stromal cells to induce osteoclastogenesis. Taken together, oral SCCs provide a suitable microenvironment for osteoclastogenesis to regulate the balance of RANKL and OPG. In this review, we introduce recent advances in the knowledge of the cellular and molecular mechanisms, by which oral SCC invades mandibular bone based on the recent findings of our lab and others.

Central glial activation mediates cancer-induced pain in a rat facial cancer model.

Peripheral and central glial activation plays an important role in development of pain hypersensitivity induced by inflammation and nerve injury. However, the involvement of glial cells in cancer pain is not well understood. The present study evaluated the peripheral and central glial activation and the effect of an inhibitor of glial activation, propentofylline, on pain-related behaviors in a rat facial cancer model of the growth of Walker 256B cells in the unilateral vibrissal pad until days 3-4 post-inoculation. As compared with sham animals, the facial grooming period was prolonged, the withdrawal latency to radiant heat stimulation was shortened, and the withdrawal threshold by von Frey hair stimulation was decreased at the inoculated region, indicating the development of spontaneous pain, thermal hyperalgesia and mechanical allodynia. In immunostainings for Iba1 and glial fibrillary acidic protein (GFAP), although there were no morphological changes of GFAP-immunopositive satellite glial cells in the trigeminal ganglion, Iba1-immunopositive microglia and GFAP-immunopositive astrocytes in the medullary dorsal horn showed large somata with cell proliferation. After the daily i.p. administration of propentofylline beginning pre-inoculation, the central glial activation was attenuated, the prolonged facial grooming was partially suppressed, and the induced allodynia and hyperalgesia from day 2 were prevented, without a change in tumor size. These results suggest that glial activation in the CNS, but not in the peripheral nervous system, mediates the enhancement of spontaneous pain and the development of allodynia and hyperalgesia at an early stage in the facial cancer model.

Differences between orofacial inflammation and cancer pain.

Rat models of orofacial cancer exhibit both allodynia and hyperalgesia; however, it is unclear whether cancer-induced pain is secondary to cancer-induced inflammation. To address this question, we compared the effects of an anti-inflammatory drug, indomethacin, on pain and neurochemical changes in the medullary dorsal horn in orofacial inflammation and cancer models. Daily peripheral administration of indomethacin largely suppressed mechanical allodynia and thermal hyperalgesia in the inflammation model. The same procedure suppressed allodynia and hyperalgesia in the cancer model, but the suppression was weak when compared with that in the inflammation model. In the medullary dorsal horn, calcitonin gene-related peptide and substance P levels were significantly increased in the inflammation model, but did not change in the cancer model. These results suggest that pain in the orofacial cancer model is not significantly mediated by cancer-induced peripheral inflammation, although it may have some involvement.

Galanin inhibits neural activity in the subfornical organ in rat slice preparation.

The activation of the subfornical organ (SFO), a circumventricular organ, induces water intake and vasopressin release. Since central administrations of galanin (GAL) suppress water intake and vasopressin release, GAL may inhibit the neural activity of SFO neurons. In the present study, we investigated effects of GAL on the SFO using molecular biological, electrophysiological and anatomical techniques. Reverse transcription-polymerase chain reaction analysis demonstrated the presence in the SFO of rats of the mRNAs for each of the three known GAL receptor subtypes (GalR1, GalR2 and GalR3). In extracellular recordings in SFO slice preparations, GAL dose-dependently inhibited the neural activity of cells from a number of recording sites. Many GAL-sensitive SFO neurons showed excitatory responses to angiotensin II (ANGII). The GalR1 agonist M617 inhibited the activity of SFO neurons, whereas the GalR2 and GalR3 agonist GAL(2-11) had almost no effect. In patch-clamp recordings, GAL induced an outward current in SFO neurons without influencing synaptic currents. An immunoelectron microscopic study revealed the existence of GAL-containing synaptic vesicles in the SFO. These results suggest that the SFO has neural inputs involving GAL. The response to GAL is inhibitory, mediated at least in part by GalR1 and provides a plausible explanation for the opposite effects of ANGII and GAL seen in vivo on water intake and vasopressin release.

[Involvement of peripheral 5-HT2A receptor activation in inflammatory pain].

Serotoninergic neuron is concerned as an one of the pivotal factor of the tissue inflammation which lead to pain behavior. Sarpogrelate HCl is a novel compound which may modulate inflammatory reaction through 5-HT2A receptor antagonist. We show the involvement of the 5-HT2A receptor activity in the inflammatory and neuropathic pain. Systemic or local sarpogrelate administration provokes antinociceptive effect on 5-HT- or formalin-produced inflammatory pain behaviour in relation to spinal glutamate. Local sarpogrelate inhibits apoptosis and neuronal degeneration after chronic construction injury(CCI). These effects are reversed by 5-HT2A receptor agonist. These results suggests sarpogrelate has a beneficial effect on hyperalgesia and neuropathic pain via inhibiting 5-HT2A receptor.

Solving the binding problem of the brain with bi-directional functional connectivity.

We propose a neural network model which gives one solution to the binding problem on the basis of 'functional connectivity' and bidirectional connections. Here, 'functional connectivity' is dynamic neuronal connectivity peculiar to temporal spike coding neural networks with coincidence detector neurons. The model consists of a single primary map and two higher modules which extract two different features shown on the primary map. There exist three layers in each higher module and the layers are connected bi-directionally. An object in the outer world is represented by a 'global dynamical cell assembly' which is organized across the primary map and the two higher modules. Detailed, but spatially localized, information is coded in the primary map, whereas coarse, but spatially extracted information or globally integrated information is coded in the higher modules. Computer simulations of the proposed model show that multiple cell assemblies sharing the same neurons partially can co-exist. Furthermore, we introduce a three-dimensional J-PSTH (Joint-Peri Stimulus Time Histogram) which is capable of tracking such cell assemblies, altering its constituent neurons as in our proposed model.

MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor beta type II receptor expression in human breast cancer cells.

Transcriptional repression of the transforming growth factor (TGF)-1P type II receptor (TPRII) gene appears to be a major mechanism to inactivate TGF-beta responsiveness in many human cancers. Because histone acetylation/deacetylation plays a role in transcriptional regulation, we have examined the effect of MS-275, a synthetic inhibitor of histone deacetylase, in human breast cancer cell lines. MS-275 showed antiproliferative activity against all human breast cancer cell lines examined and induced TbetaRII mRNA, but not TGF-beta type I receptor mRNA. MS-275 caused an accumulation of acetylated histones H3 and H4 in total cellular chromatin. An increase in the accumulation of acetylated histones H3 and H4 was detected in the TbetaRII promoter after treatment with MS-275. However, the level of histone acetylation did not change in chromatin associated with the TGF-beta type I receptor gene. MS-275 treatment enhanced TGF-beta1-induced plasminogen activator inhibitor 1 expression. Thus, antitumor activity of MS-275 may be mediated in part through the induction of TbetaRII expression and consequent potentiation of TGF-beta signaling.

A novel synthetic DNA minor groove binder, MS-247: antitumor activity and cytotoxic mechanism.

PURPOSE: MS-247 is a novel synthetic compound possessing a DNA-binding moiety and a DNA-alkylating residue, chlorambucil. In this study, we evaluated the antitumor activity of MS-247 against murine tumor cell lines and its effects on DNA molecules in both cell-free and cellular systems. METHODS: The in vitro cytotoxic activity of MS-247 was evaluated against four murine tumor cell lines, P388, L1210, Colon26 and B16, and its in vivo antitumor activity was also tested in comparison with Adriamycin (ADM), cisplatin (CDDP) and paclitaxel. The ability of MS-247 to associate with the DNA minor groove was assessed by measuring quenching of Hoechst 33342 fluorescence. DNA-DNA interstrand crosslinks (ICL) were detected by an alkaline elution assay for cellular DNA and a band-shift assay using the plasmid pBR322. The effects of MS-247 on macromolecule synthesis (DNA, RNA and proteins) were examined by measuring incorporation of the radiolabeled precursors. RESULTS: MS-247 exhibited in vitro cytotoxicity with IC(50) values ranging 11 to 500 nM, and MS-247 given i.v. showed strong in vivo antitumor activity against i.p.-implanted L1210 leukemia cells and s.c.-implanted Colon26 carcinoma cells, and moderate activity against i.p.-implanted P388 leukemia cells but no apparent activity against s.c.-implanted B16 melanoma cells. MS-247 reversibly displaced Hoechst 33342 bound to DNA within a few minutes, and irreversibly formed ICL within 1-6 h in both the cell-free system and the cellular system. These results suggest that an association of MS-247 with the DNA minor groove occurred more quickly than ICL formation. The inhibition of DNA synthesis was more prominent than the inhibition of RNA and protein synthesis in L1210 cells exposed to MS-247, and a 6-h incubation with MS-247, which formed apparent ICL in the cellular system, strongly inhibited DNA synthesis. This result suggests that impairment of DNA replication preceded the inhibition of RNA and protein synthesis and that ICL formation greatly contributed to the inhibition of macromolecule synthesis. CONCLUSION: The results of this study suggest that MS-247 exerts its cytotoxic effect through impairment of DNA function by getting into the minor groove of DNA and subsequently forming ICL. MS-247 has potent antitumor activity with a different spectrum from the activity of clinically proven antitumor agents such as paclitaxel, ADM and CDDP against several murine tumor cell lines. This result suggests that MS-247 may be useful for the treatment of human cancers.

Potent antitumor activity of MS-247, a novel DNA minor groove binder, evaluated by an in vitro and in vivo human cancer cell line panel.

We synthesized a novel anticancer agent MS-247 (2-[[N-[1-methyl-2-[5-[N-[4-[N,N-bis(2-chloroethyl) amino] phenyl]] carbamoyl]-1H-benzimidazol-2-yl] pyrrol-4-yl] carbamoyl] ethyldimethylsulfonium di-p-toluenesulfonate) that has a netropsin-like moiety and an alkylating residue in the structure. We evaluated antitumor activity of MS-247 using a human cancer cell line panel coupled with a drug sensitivity database and subsequently using human cancer xenografts. The average MS-247 concentration required for 50% growth inhibition against a panel of 39 cell lines was 0.71 microM. The COMPARE analysis revealed that the differential growth inhibition pattern of MS-247 significantly correlated with those of camptothecin analogues and anthracyclins, indicating that MS-247 and the two drug groups might have similar modes of action. MS-247 exhibited remarkable antitumor activity against various xenografts. A single i.v. injection of MS-247 significantly inhibited the growth of all 17 xenografts tested, which included lung, colon, stomach, breast, and ovarian cancers. In many cases, MS-247 was more efficacious than cisplatin, Adriamycin, 5-fluorouracil, cyclophosphamide, VP-16, and vincristine and was almost comparable with paclitaxel and CPT-11; these are the most clinically promising drugs at present. MS-247 was noticeably more effective than paclitaxel (in HCT-15) and CPT-11 (in A549, HBC-4, and SK-OV-3). The toxicity of MS-247, indicated by body weight loss, was reversible within 10 days after administration. The MS-247 mode of action showed DNA binding activity at the site where Hoechst 33342 bound, inhibited topoisomerases I and II (as expected by the COMPARE analysis) blocked the cell cycle at the G2-M phase, and induced apoptosis. These results indicate that MS-247 is a promising new anticancer drug candidate to be developed further toward clinical trials.

Synthesis and histone deacetylase inhibitory activity of new benzamide derivatives.

Newly synthesized benzamide derivatives were evaluated for their inhibitory activity against histone deacetylase. The structure of these derivatives was unrelated to the known inhibitors, and IC(50) values of the active compounds were in the range of 2-50 microM. Structure-activity relationship on the benzanilide moiety showed that the 2'-substituent, an amino or hydroxy group, was indispensable for inhibitory activity. Although the electronic influence of the substituent in the anilide moiety showed only a small effect on inhibitory activity, the steric factor in the anilide moiety, especially at positions 3'and 4', played an important role in interaction with the enzyme. Among these benzamide derivatives, MS-275 (1), which showed significant antitumor activity in vivo, has been selected for further investigation.

Nerve growth factor inducer, 4-methyl catechol, potentiates central sensitization associated with acceleration of spinal glutamate release after mustard oil paw injection in rats.

1. In rats, injection of mustard oil (MO) into the paw caused a gradual increase in flinching of the injected paw and this algogenic behavior corresponded with an increase in the CSF-Glu level. 2. The nerve growth factor (NGF) inducer, 4-methyl catechol (4MC), enhanced the frequency of flinching and this effect was dose dependent. In addition, spinal CSF-Glu release was significantly above baseline 10 min after MO injection. In contrast, morphine (MOR) pretreatment completely blocked this behavioral and neurohumoral effect. 3. Anti-NGF paw injection attenuated the algogenic behavior and spinal Glu release otherwise observed after 4MC treatment. 4. The results demonstrated that MO-induced hyperalgesia is associated with increased CSF-Glu release and that this effect is potentiated by a NGF inducer. These data also suggest a possible involvement of NGF in the development of central sensitization after acute peripheral nociceptive stimulation.

A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors.

Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21(WAF1/CIP1) and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21(WAF1/CIP1) tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.

Increase in the threshold of pain and touch sensation in the human face with clonidine plus 30% nitrous oxide.

OBJECTIVE: This study was undertaken to assess the effects of clonidine combined with 30% nitrous oxide on tactile and pain sensations in the human face. STUDY DESIGN: Thirty-three subjects were involved in the study. The subjects were divided into 4 groups: 100% oxygen with placebo; 30% N2O with placebo; 100% oxygen with clonidine (0.075 mg), and 30% N2O with clonidine. Three tests for the threshold of pain sensation and tactile sensation were made at 60 minutes before and 0, 15, and 30 minutes during N2O or O2 inhalation. RESULTS: (1) The N2O with clonidine significantly increased the threshold of pain and tactile sensation in comparison with the other 3 treatments. (2) In terms of pain sensation, both N2O and clonidine showed significant increases in threshold of pain in comparison with the control values. CONCLUSIONS: These results indicate that the analgesic effects of 30% nitrous oxide are enhanced when use of the gas is combined with prior clonidine administration.

Modulation of formalin-evoked hyperalgesia by intrathecal N-type Ca channel and protein kinase C inhibitor in the rat.

1. omega-CgTx attenuated formalin-evoked biphasic flinches, while PKC inhibitor (STU) attenuated phase 2 and was reversed by PDBu. 2. omega-CgTx and STU suppressed the increase in CSF-glutamate after formalin injection. 3. Morphine completely suppressed both increased flinching and CSF glutamate release. 4. Thus, omega-CgTx (N-type Ca channels) may regulate neurotransmitter release evoked by C fiber activation and the formalin-evoked hyperalgesia may possibly be provoked as a result of PKC activation elicited by both presynaptic neurotransmitter release and activation of NMDA receptors in the spinal neurons.

Central noradrenergic mediation of nitrous oxide-induced analgesia in rats.

PURPOSE: Although several studies have demonstrated that both supra opiate receptors and spinal alpha 2 adrenoceptors play a mediating role in nitrous oxide(N2O) analgesia, controversy still exists. The present study was undertaken to evaluate further the involvement of noradrenergic (NA) neuronal activity in N2O analgesia by investigating tail-flick latency and supra- and spinal NA levels in rats. METHODS: In an analgesia study, effect of N2O 75% and its modification were evaluated using the tail-flick test in male Wistar rats. Results were expressed as % maximum possible effect (MPE). Modification of N2O analgesia was examined in rats pretreated with either the alpha 2 receptor agonist, clonidine(CLO: 150 micrograms.kg-1, i.p.), alpha 2 receptor antagonist, idazoxone(IDZ: 100 micrograms.kg-1, i.v.) by lesioning the locus coeruleus (LC) seven days before exposure to N2O, or naloxone (5 mg.kg-1, i.v.). Also, in a NAergic neuronal transmission study, the changes in NA content at LC and spinal cord were determined using HPLC-ECD. RESULTS: Nitrous oxide produced analgesia, % MPE increased to a maximum of 78% at 30 min, thereafter declining to 38% at 120 min. Clonidine potentiated the analgesic effect of N2O at 120 min (80%). The analgesic effect of N2O was attenuated by IDZ or by LC lesioning. However, naloxone, in a dose sufficient to block morphine-induced analgesia, had no effect. With N2O exposure, NA content was decreased by 52% in the LC and by 20% at spinal cord. With morphine, NA content did not differ from the control group. CONCLUSION: The data suggest that N2O-induced analgesia is principally mediated by activation of the descending inhibitory NAergic system and/or increased NA release at spinal cord which may lead to presynaptic inhibition of primary afferent neurotransmitter release and hyperpolarize the dorsal horn neurons by alpha 2 receptors.

Inhibition of cerebral metabolic and circulatory responses to nitrous oxide by 6-hydroxydopamine in dogs.

PURPOSE: To determine whether cerebral metabolic and circulatory consequences of N2O result from activation of the sympathoadrenal system. The effects of pretreatment with intracisternal injection of 6-OHDA, which produces chemical sympathectomy, were studied in dogs. METHOD: Seven days before measurement dogs were pretreated with intracisternal injection of either saline vehicle (sham-group) or 100 micrograms.kg-1 6-hydroxydopamine (6-OHDA, group). Cerebral blood flow (CBF) was measured using an electromagnetic flow-meter probe and cerebral metabolic rate for oxygen (CMRO2) was calculated as the product of CBF and arterial-sagittal sinus blood oxygen content difference [C(a-v)O2]. RESULTS: In the sham group, N2O (60%) increased CMRO2 from 6.11 +/- 0.21 ml.100 g-1.min-1 to 7.10 +/- 0.39 ml.100 g-1.min-1 and CBF from 63 +/- 5 ml.100 g-1.min-1 to 173 +/- 26 ml.100 g-1.min-1. In the 6-OHDA group, CMRO2, did not change during N2O exposure, whereas CBF increased from 61 +/- 3 ml.100 g-1.min-1 to 135 +/- 19 ml.100 g-1.min-1 but less then in the sham group. The 6-OHDA group displayed a reduction in cortical noradrenaline (NA) concentration from 263.2 +/- 35.6 ng.g-1 to 102.7 +/- 16.5 ng.g-1. Cortical dopamine (DA) concentration was not affected by 6-OHDA administration. CONCLUSION: These results suggest that most of the increase in CMRO2 and at least a part of the increase in CBF during N2O exposure in the sham-group are related to sympathoadrenal-stimulating effects of N2O.

Characterization of heat-hyperalgesia in an experimental trigeminal neuropathy in rats.

Secondary trigeminal neuralgia (STN) follows an injury to the trigeminal nerve or one of its branches. Although rare, this condition results in great suffering and it is notoriously difficult to treat. The experimental analysis of painful neuropathy due to damage to the innervation of the limbs (e.g., the sciatic nerve) has progressed rapidly in recent years, but very few reports have appeared concerning experimental neuropathy in the trigemenial region. We report here an experimental rat model of trigeminal neuropathic pain produced by a chronic constriction injury to the infraorbital nerve (CCI-ION), and on a method that detects heat-evoked pain-related behavior. Rats with the CCI-ION have clear signs of heat-hyperalgesia when stimulated on the snout (the vibrissal pad). The hyperalgesia is seen both ipsi- and contralateral to the side of nerve injury, but is significantly more severe ipsilaterally, and lasts about 12 days.

The Ca2+-dependent binding of calmodulin to an N-terminal motif of the heterotrimeric G protein beta subunit.

Ca2+ ion concentration changes are critical events in signal transduction. The Ca2+-dependent interactions of calmodulin (CaM) with its target proteins play an essential role in a variety of cellular functions. In this study, we investigated the interactions of G protein betagamma subunits with CaM. We found that CaM binds to known betagamma subunits and these interactions are Ca2+-dependent. The CaM-binding domain in Gbetagamma subunits is identified as Gbeta residues 40-63. Peptides derived from the Gbeta protein not only produce a Ca2+-dependent gel mobility shifting of CaM but also inhibit the CaM-mediated activation of CaM kinase II. Specific amino acid residues critical for the binding of Gbetagamma to CaM were also identified. We then investigated the potential function of these interactions and showed that binding of CaM to Gbetagamma inhibits the pertussis toxin-catalyzed ADP-ribosylation of Galphao subunits, presumably by inhibiting heterotrimer formation. Furthermore, we demonstrated that interaction with CaM has little effect on the activation of phospholipase C-beta2 by Gbetagamma subunits, supporting the notion that different domains of Gbetagamma are responsible for the interactions of different effectors. These findings shed light on the molecular basis for the interactions of Gbetagamma with Ca2+-CaM and point to the potential physiological significance of these interactions in cellular functions.

Effects of midazolam on pain sensations in the face.

OBJECTIVES: This study investigated the effects of midazolam, a sedative, on tactile and pain sensations on the skin of the chin. STUDY DESIGN: Thirty-seven volunteers were segregated into four groups; the first group was the control group; the second to fourth groups were administered 0.025 mg/kg, 0.05 mg/kg, and 0.075 mg/kg of midazolam, respectively, as a bolus injection. All volunteers reclined in a dental chair for the experiment. Tactile and pain sensations were determined over time after injection of midazolam, the former using von Fray thread, the latter using an esthesiometer. RESULTS: Thresholds of tactile sensitivity and of pain were statistically significantly different from control values at 10 minutes after injection of midazolam in the 0.05 mg/kg group and in the 0.075 mg/kg group. CONCLUSION: Although 0.025 mg/kg of midazolam produced sedation, at least 0.05 mg/kg of this agent was required to alter the thresholds for perception of tactile and painful stimulation.