NIH Swiss Mice Research Articles

Effect of hyperbaric oxygen (HBO 2 ) treatment on the expression of neuronal nitric oxide synthase (nNOS) in the mouse brain

Treatment with hyperbaric oxygen (HBO2) has been demonstrated by our laboratory and others (Thom and Buerk, Adv. Exp. Med. Biol. 510:133, 2003; Ohgami et al., Brain Res. Bull. 75:668, 2008) to stimulate the production of nitric oxide (NO). The aim of this study was to determine whether exposure to HBO2 increases expression of nNOS enzyme in different regions of the mouse brain. Male NIH Swiss mice were exposed to HBO2 at 3.5 ATA for 60 min then killed at 2, 4, 6 and 8 hr post-HBO2. The brains were removed and sectioned using a mouse brain matrix to make six 2 mm-thick coronal sections. Extracts of each section were analyzed by immunoblotting detection of nNOS. Quantification of nNOS expression levels was achieved by measuring the near infrared fluorescence detection of nNOS protein bands. The results demonstrate that the nNOS levels of HBO2-treated mice were higher than that of room air-exposed control mice, especially in more caudal regions of the brain, and peaked at 4 hrs post-HBO2. At 8 hr post-HBO2, levels of nNOS expression were comparable to that of control mice. Based on these results, we conclude that HBO2 treatment increases the expression of nNOS in the mouse brain. (This research was supported by NIH Grant GM-77153, the Allen I White Distinguished Professorship and an Institutional SURF Award from ASPET.)

Involvement of a NO–cyclic GMP–PKG signaling pathway in nitrous oxide-induced antinociception in mice

The antinociceptive effect of nitrous oxide (N 2O) is dependent on nitric oxide (NO); however, the next step in the pathway activated by NO is undetermined. The present study was conducted to test the hypothesis that a N 2O action involves sequential activation of NO synthase, soluble guanylyl cyclase and protein kinase G to induce an antinociceptive effect in mice. The antinociceptive responsiveness of male NIH Swiss mice to N 2O was assessed using the acetic acid abdominal constriction test. Different groups of mice were pretreated with either saline, the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO), the guanylyl cyclase-inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), the protein kinase G-inhibitor Rp-isomer of 8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS) or the selective phosphodiesterase V-inhibitor 1,2-dihydro-2-[(2-methyl-4-pyridinyl)methyl]-1-oxo-8-(2-pyrimidinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T 0156). Vehicle (saline)-pretreated mice responded to N 2O in a concentration-dependent manner. This antinociceptive effect was antagonized by systemic pretreatment with carboxy-PTIO and ODQ and central pretreatment with Rp-8-pCPT-cGMPS. In each case, the dose–response curve for N 2O was progressively shifted to the right by increasing the dose of each pretreatment drug. On the other hand, N 2O-induced antinociception was enhanced by systemic pretreatment with T 0156; the dose–response curve for N 2O was shifted to the left. The ATP-sensitive potassium channel blocker glibenclamide was without influence on the antinociceptive effect of N 2O. These results support the hypothesis that N 2O-induced antinociception in mice is mediated by a NO–cyclic GMP–PKG pathway.

The acute antinociceptive effect of HBO 2 is mediated by a NO–cyclic GMP–PKG–K ATP channel pathway in mice

Previous research has found that hyperbaric oxygen (HBO 2) produces an acute antinociceptive effect that is dependent on nitric oxide (NO). The present study was undertaken to determine whether HBO 2-induced acute antinociception might involve a NO–cyclic GMP–protein kinase G–ATP-sensitive potassium (K ATP) channel pathway. Male NIH Swiss mice were subjected to a 5-min HBO 2 treatment (100% oxygen at 3.5 absolute atmospheres) and antinociception was assessed over the next 6 min still under HBO 2 using the acetic acid abdominal constriction test. Pretreatment with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO, an NO scavenger), 1H-[1,2,4]-oxadiazolo-[4,3-a]quinoxalin-1-one) (a soluble guanylyl cyclase-inhibitor, Rp-8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate (a protein kinase G-inhibitor) or glibenclamide (an ATP-sensitive potassium channel-inhibitor) all led to antagonism of the HBO 2-induced acute antinociception in a dose-dependent manner. These findings suggest that HBO 2-induced acute antinociception might be due to activation of a NO–cyclic GMP–protein kinase G–K ATP channel pathway.

A new biodosimetric method: branched DNA-based quantitative detection of B1 DNA in mouse plasma

A simple and accurate method for measuring the biological effects of radiation is of increasing importance, especially in mass casualty scenarios. We have therefore developed a new biodosimetric technique targeting circulating B1 DNA in mouse plasma by branched DNA signal amplification for rapid quantification of plasma DNA. This technology targets repetitive elements of the B1 retrotransposon in the mouse genome, followed by signal amplification using Panomics Quantigene 2.0 reagents. Evaluation was conducted concerning precision, accuracy and linearity. Plasma samples were collected from mice 0-24 h after 0-10 Gy total body irradiation (TBI). The average inter- and intra-assay coefficients of variance were 8.7% and 12.3%, respectively. The average recovery rate of spiked DNA into plasma was 89.5%. This assay revealed that when BALB/c and NIH Swiss mice were exposed to 6 Gy TBI, plasma B1 DNA levels increased significantly at 3 h post-TBI, peaked at 9 h and gradually returned toward baseline levels in 24 h. A dose-dependent change in plasma DNA was observed at 9 h post-TBI; the dose-response relation was monotonic, exhibiting linearity for BALB/c mice from 3 to 6 Gy (r = 0.993) and NIH Swiss mice from 3 to 7 Gy (r = 0.98). This branched DNA-based assay is reliable, accurate and sensitive in detecting plasma B1 DNA quantitatively. A radiation dose-correlated increase in plasma B1 DNA was demonstrated in BALB/c and NIH Swiss mice in the dose range from 3 to 6 Gy, suggesting that plasma B1 DNA has potential as a biomarker for radiation biological effect.

Seizure tests distinguish intermittent fasting from the ketogenic diet

Calorie restriction can be anticonvulsant in animal models. The ketogenic diet was designed to mimic calorie restriction and has been assumed to work by the same mechanisms. We challenged this assumption by profiling the effects of these dietary regimens in mice subjected to a battery of acute seizure tests. Juvenile male NIH Swiss mice received ketogenic diet or a normal diet fed in restricted quantities (continuously or intermittently) for ∼12 days, starting at 3-4 weeks of age. Seizures were induced by the 6 Hz test, kainic acid, maximal electroshock, or pentylenetetrazol. The ketogenic and calorie-restricted diets often had opposite effects depending on the seizure test. The ketogenic diet protected from 6 Hz-induced seizures, whereas calorie restriction (daily and intermittent) increased seizure activity. Conversely, calorie restriction protected juvenile mice against seizures induced by kainic acid, whereas the ketogenic diet failed to protect. Intermittent caloric restriction worsened seizures induced by maximal electroshock but had no effect on those induced by pentylenetetrazol. In contrast to a longstanding hypothesis, calorie restriction and the ketogenic diet differ in their acute seizure test profiles, suggesting that they have different underlying anticonvulsant mechanisms. These findings highlight the importance of the 6 Hz test and its ability to reflect the benefits of ketosis and fat consumption.

Abstract B50: A new class of diazonamide analogs with improved antitumor activity

Abstract Diazonamide A is a natural product originally isolated from a marine organism. It potently induces G2/M cell cycle arrest. Its molecular mode of action is not fully understood but its potential as a cancer therapeutic is high. We previously reported that AB5, a close analog of diazonamide A, exhibits potent anti-tumor activity in mouse xenograft models. Tumor regression occurs in the absence of systemic toxicity typical of anti-mitotic chemotherapy. Here we show for the first time that the natural diazonamide compound possesses strong tumor killing activity in vivo xenograft models. To streamline our diazonamide synthesis and to define a minimal pharmacophore, we prepared a series of truncated structures. One of these, JP1838, was found to be as potent as AB5. Its simplified structure and shortened synthesis allowed for extensive structure activity relationship analyses. Several modifications markedly improved activity. One analog, JP2295, is >20 fold more potent than AB5 in a MIA Paca-2 pancreatic cancer xenograft model. More importantly, in HPAC pancreatic cancer and HCT-116 colon cancer xenograft models, JP2295 demonstrates greater efficacy and less toxicity than docetaxel and vinorelbine. Safety studies were therefore carried out in NIH Swiss mice. At doses10-fold higher than required for strong efficacy, JP2295 does not exhibit toxicities common to docetaxel and vinorelbine treatments. These include body weight loss, neutropenia, and myelosuppression. Such a large therapeutic window is unique for a small molecule antimitotic and suggests further development is warranted. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B50.

Suppression of aberrant transient receptor potential cation channel, subfamily V, member 6 expression in hyperproliferative colonic crypts by dietary calcium.

Dietary calcium is believed to reduce colon cancer risk, but the mechanism by which this occurs is poorly understood. Employing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we previously showed that a high-calcium diet (hCa) significantly abrogated hyperplasia in the distal colons of NIH-Swiss mice. Here, we explored the mechanism of dietary protection by hCa by analyzing the expression of genes involved in the regulation of Ca uptake/flux in the intestinal epithelium, including the Ca-sensing receptor, vitamin D receptor, Ca binding protein, and transient receptor potential cation channels, subfamily V, members 5 and 6 (TRPV5/6). Interestingly, while TRPV6 expression increased significantly during TMCH, the expression of the other gene products was unchanged. This elevated TRPV6 expression was significantly abrogated by a hCa diet. Immunofluorescence revealed apical membrane localization of TRPV6 in the normal colon, whereas during TMCH we observed intense apical pole and cytoplasmic staining along the entire longitudinal crypt axis, including the expanded proliferating zone. The hCa diet reversed this effect. In humans, overexpression of TRPV6 was associated with early-stage colon cancer, and in colon carcinoma cells, inhibition of TRPV6 expression by small interfering RNA inhibited their proliferation and induced apoptosis. TRPV6 small interfering RNA also diminished the transcriptional activity of the calcium-dependent nuclear factors in activated T cells. Thus the aberrant overexpression of TRPV6 contributes to colonic crypt hyperplasia in mice and to colon cancer cell proliferation in humans. Therefore, it is likely that suppression of TRPV6 by a hCa diet is required for its protective effects in the colon.

Tumors Induced in Mice by Direct Inoculation of Plasmid DNA Expressing Both Activated H-ras and c-myc

Vaccines contain residual DNA derived from the cells used to produce them. As part of our investigation to assess the risk of this cellular DNA, we are developing a quantitative in vivo assay to assess the oncogenicity of DNA. In an earlier study, we had generated expression plasmids for two oncogenes--human activated T24-H-ras and murine c-myc--and had shown that these two plasmids, pMSV-T24-H-ras and pMSV-c-myc, could act in concert to induce tumors in mice, although the efficiency was low. In this study, we took two approaches to increase the oncogenic efficiency: 1) both oncogene-expression cassettes were placed on the same plasmid; 2) transfection facilitators, which increase DNA uptake and expression in vitro, were tested. The dual-expression plasmid, pMSV-T24-H-ras/MSV-c-myc, is about 20-fold more efficient at tumor induction in newborn NIH Swiss mice than the separate expression plasmids, with tumors being induced with 1 microg of the dual-expression plasmid DNA. However, none of the transfection facilitators tested increased the efficiency of tumor induction. Based on these data, the dual-expression plasmid pMSV-T24-H-ras/MSV-c-myc will be used as the positive control to develop a sensitive and quantitative animal assay that can be used to assess the oncogenic activity of DNA.

Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells

Fibroblasts are sentinel cells that could serve as intermediaries in the immune reaction in the inflammatory process. In this work, we investigate the action of the muscarinic agonist carbachol (CARB) on the expression and function of nitric oxide synthase (NOS) and cyclooxygenase (COX) in fibroblasts under normal or inflammatory conditions. The normal fibroblast cell line, 3T3, from NIH swiss mouse embryo, was used. The inflammatory milieu was mimicked with lipopolysaccharide (LPS) (10 ng/ml) plus interferon gamma (IFNgamma) (0.5 ng/ml). Nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production were measured by Griess reagent and radioimmunoassay, respectively. NOS, COX, and nuclear transcription factor kappa B (NF-kappaB) were studied by Western blot. CARB increased NO synthesis by 57 +/- 5%, while a 150 +/- 10% increase in NO liberation was triggered by LPS plus IFNgamma treatment. CARB added to LPS plus IFNgamma potentiated NO synthesis by 227 +/- 19%. CARB also upregulated NOS1 protein expression via NF-kappaB activation. In addition CARB and LPS plus IFNgamma stimulated PGE(2) synthesis by 72 +/- 9 and 42 +/- 4%, respectively, while CARB added to LPS plus IFNgamma treated cells produced a synergism in PGE(2) liberation (130 +/- 12%) via COX-2. Activation of muscarinic acetylcholine receptors can mimic mild inflammatory conditions or can deepen pre-existing inflammation, establishing a fine-tuned set-up on fibroblasts that in turn could be alerting the immune system.

Antagonism of the antinociceptive effect of nitrous oxide by inhibition of enzyme activity or expression of neuronal nitric oxide synthase in the mouse brain and spinal cord

Previous studies have implicated nitric oxide (NO) in the antinociceptive response to the anesthetic gas nitrous oxide (N 2O). The present study was conducted to confirm this NO involvement using pharmacological and gene knockdown and knockout strategies to inhibit the supraspinal and spinal production of NO. Antinociceptive responsiveness to 70% N 2O was assessed using the acetic acid (0.6%) abdominal constriction test in NIH Swiss mice following intracerebroventricular (i.c.v.) or intrathecal (i.t.) pretreatment with the NOS-inhibitor l-N G-nitro arginine methyl ester (L-NAME) or an antisense oligodeoxynucleotide (AS-ODN) directed against neuronal NOS (nNOS). Experiments were also conducted in mice homozygous for a defective nNOS gene (nNOS −/−). Mice that were pretreated i.c.v. or i.t. with L-NAME (1.0 µg) both exhibited 80–90% reduction in the magnitude of the N 2O-induced antinociceptive response. Mice that were pretreated i.c.v. or i.t. with nNOS AS-ODN (3 × 25 µg) exhibited a 60–80% antagonism of the antinociceptive response. Compared to wild-type mice, nNOS knockout mice showed a 60% reduction in N 2O-induced antinociception. These findings consistently demonstrate that transient or developmental suppression of nNOS expression significantly reduces antinociceptive responsiveness to N 2O. NO of both supraspinal and spinal origin, therefore, plays an important role in the antinociceptive response to N 2O.

Genetically inbred Balb/C mice are more sensitive to an effect of flurazepam and more resistant to an effect of stress than a genetically outbred mouse strain

The inbred Balb/c mouse strain was more sensitive than the outbred NIH Swiss mouse to flurazepam’s ability to antagonize electrically precipitated seizures. In prior work, a reduction in flurazepam’s antiseizure efficacy was not observed 24 h after forcing Balb/c mice to swim for up to 10 min in ambient temperature water. Thus, we wondered if a stress-induced reduction would be observed after forcing mice to swim for up to 10 min in cold (6 °C) water, a more severe stress. The current data show that 24 h after exposure to this stress, the ability of flurazepam to raise the threshold voltage for the elicitation of tonic hindlimb extension in the Balb/c mouse strain was reduced. The genetically inbred Balb/c mouse strain is emerging as an interesting animal model in which to study interactions of stress and genetic factors that affect endogenous neurotransmission mediated by l-glutamate and GABA at the NMDA and GABA A receptor complexes, respectively.

Differential involvement of 5‐hydroxytryptamine1A (5‐HT1A) receptors in the antinociceptive effects of hyperbaric oxygen (HBO2) and nitrous oxide (N2O) in mice

Earlier we reported that a 60‐min exposure to HBO2 induced an antinociceptive response that lasted for up to 90 min following exposure [Zylstra et al., FASEB J 22:711.17, 2008]. Recent studies have reported a sustained antinociceptive effect of a selective, high‐efficacy 5‐HT1A receptor agonist in rodents [Deseure et al., Eur J Pharmacol 568:134, 2007]. To determine whether supraspinal or spinal 5‐HT1A receptors might be involved in HBO2‐ or N2O‐induced antinociception, male NIH Swiss mice (20‐30 g) were exposed to HBO2 (2.5 ATA, 60 min) then removed to room air for 90 min or N2O acutely (11 min). Mice were pretreated with intracerebroventricular (i.c.v.) or intrathecal (i.t.) selective 5HT1A antagonist WAY‐100635 prior to antinociceptive testing using the abdominal constriction test. Results show that both i.c.v. and i.t. pretreatments with WAY‐100635 produced comparable and dose‐related antagonism of the antinociceptive response to HBO2. On the other hand, i.c.v. and i.t. pretreatment with WAY‐100635 had no effect on N2O‐induced antinociception. These results suggest that both supraspinal and spinal 5HT1A receptors are involved in the 90‐min antinociceptive response to a 60‐min exposure to HBO2. However, there does not appear to be a role for 5HT1A receptors in the antinociceptive response to N2O. (Supported in part by NIH Grant GM‐77153, the College of Pharmacy and the Chico Hyperbaric Center.)

Hyperbaric oxygen (HBO2) induces a two‐phase antinociceptive response of unusually long duration in mice

HBO2 is approved by the FDA for limited clinical indications but not for chronic pain. Earlier we showed that HBO2 treatment produces an antinociceptive effect mediated by nitric oxide (NO) and opioid mechanisms [Zylstra et al., FASEB J 22, 711.16 & 711.17, 2008]. In this study, we assessed the antinociceptive effect of multiple HBO2 treatments. Male NIH Swiss mice (20‐30 g) were exposed to HBO2 at 2.5 ATA for 4×60‐min then returned to their home cages for different time intervals before testing with the acetic acid‐induced abdominal constriction test. Results revealed a robust antinociception of at least 6 hr after the last HBO2 treatment. This response was followed a day later by an emerging and equally robust late‐phase antinociceptive effect that surprisingly lasted for two weeks following the last HBO2 treatment. The early phase was antagonized by pretreatment with naltrexone (NTX) and the late phase was antagonized by the selective 5‐HT1A antagonist WAY‐100635 but not NTX. Continuous treatment with NTX or the NO synthase‐inhibitor L‐NAME during the 4 days of HBO2 treatment also reduced the late‐phase response. Based on these findings, we suggest that the early‐phase antinociception involves an opioid pathway, while the longer‐acting late‐phase antinociception involves an NO‐opioid‐5‐HT1A pathway. (Supported in part by NIH Grant GM‐77153, the College of Pharmacy and the Chico Hyperbaric Center.)

Store operated calcium entry in NIH-3T3 cells

)bythe patch clamp technique. Therefore, thapsigargin-induced SOCE is a useful approach to investigatemechanisms of SOCE.Immortal NIH3T3 cells have been establishedfrom the embryonic NIH Swiss mice fibroblasts. TheNIH-3T3 cell is a useful cell line for investigationof transformation, stress fiber formation, or feedercells of keratinocytes. NIH-3T3 cells have epidermalgrowth factor (EGF) receptors (2), fibroblast growthfactor (FGF) receptors (3), platelet derived growthfactor (PDGF) receptors (3), bombesin receptors(3) bradykinin receptors (4) and ATP receptors(5), all of those receptors require calcium influx.Stimulation of those receptors mobilizes stored Ca

Expression of NR1, NR2A and NR2B NMDA receptor subunits is not altered in the genetically-inbred Balb/c mouse strain with heightened behavioral sensitivity to MK-801, a noncompetitive NMDA receptor antagonist

The genetically-inbred Balb/c mouse strain shows heightened sensitivity to the ability of MK-801 (dizocilpine), a noncompetitive NMDA receptor antagonist, to raise the threshold voltage necessary to precipitate tonic hindlimb extension and elicit irregular episodes of intense jumping behavior (referred to as “popping”), relative to other inbred mouse strains and the outbred NIH Swiss mouse. Moreover, an allosteric modulatory effect of sarcosine, a glycine reuptake inhibitor, on MK-801′s antagonism of electrically precipitated seizures was detected 24 h after Balb/c mice were forced to swim in cold water for up to 10 min; this was not observed in unstressed Balb/c mice or stressed or unstressed NIH Swiss mice. Phencyclidine (PCP), a noncompetitive NMDA receptor antagonist that binds to the same hydrophobic channel domain as MK-801, precipitates a schizophreniform psychosis in susceptible individuals that shares descriptive similarities with schizophrenia. This observation has led to the hypothesis that NMDA receptor hypofunction (NRH) is involved in the pathophysiology of schizophrenia and the testing of pharmacotherapeutic strategies to facilitate NMDA receptor-mediated neurotransmission in patients with this disorder (e.g., glycine reuptake inhibitors). The heightened behavioral sensitivity of the Balb/c mouse to MK-801 suggests that this mouse strain may be a useful model to study “psychosis-proneness” and screen for positive allosteric modulators of NMDA receptor-mediated neurotransmission. Conceivably, strain differences in the pharmacology of the NMDA receptor are due to differences in the relative expression of individual NMDA receptor subunits to each other (i.e., combinatorial regulation). The current study compared the normal protein expression patterns of six of the eight identified splice variant isoforms of the NR1 NMDA receptor subunit, and NR2A and NR2B subunits in the hippocampus and cerebral cortex of Balb/c and NIH Swiss mice. The heightened behavioral sensitivity of the Balb/c genetically-inbred mouse strain to MK-801, compared to the outbred NIH Swiss mouse strain, does not appear to result from relative alterations of expression of these NMDA receptor protein subunits that were examined.

Antigen(s)-specific tumour-infiltrating lymphocytes from tumour induced by human herpes virus-6 (HHV-6) DNA transfected NIH 3T3 transformants

Tumour infiltrating lymphocytes (TIL) have recently been shown to mediate potent therapeutic effects in certain malignancies in mice and in humans. To understand the mechanism of TIL immunotherapy it would be advantageous to generate tumour-specific TIL and to study a defined system of TIL and target cells in which the tumour epitope(s) recognized by TIL might be identified. We have established tumourigenic cell lines by transfection of NIH 3T3 cells with the entire genome of human herpesvirus-6 (HHV-6) and its small fragment (about 5% of the viral DNA sequence). Injection of these cells into nude mice produced tumours termed G-2T and 14-2T, respectively. Cell lines derived from these tumours when injected in NIH Swiss mice produced tumours, G-2TS and 14-2TS, respectively. We have generated TIL from G-2TS tumour that can kill G-2TS tumour cells in vitro but not other related tumours (14-2TS or MCA-106). These TIL can be expanded between 2-6.5 every 3-5 days. The TIL proliferated in tissue culture in response to recombinant interleukin-2 and interleukin-4 and maintained their tumor specificity for up to 6 months in vitro. Their phenotype was Thy 1.2+, Lyt-2+ and L3T4-. The availability of such tumour-specific stable TIL lines and specific viral-transformed targets will provide an opportunity to characterize the tumour-associated antigen critical for the specific cytotoxicity in this system and thereby to clarify the mechanism of this promising immunological approach to cancer therapy.

Role of nitric oxide (NO) in the hyperbaric oxygen (HBO2)‐induced long‐lasting activation of endogenous opioid systems in mice

Exposure to HBO2 causes a long‐lasting analgesic effect in patients with chronic pain (Kiralp et al., J. Int. Med. Res. 32:258–262, 2004). To determine the duration of antinociceptive effect in an animal model following exposure to HBO2, male NIH Swiss mice (20–30 g) were exposed to HBO2 at 2.5 ATA for 60 min then removed to room air for varying durations of time (5, 15, 30, 60, 90, 120, 150 or 180 min) prior to antinociceptive testing. The antinociceptive effect of HBO2 remained robust up to 90 min but subsided by 120 min. By contrast, mice exposed to 70% nitrous oxide for 60 min exhibited antinociception only through the 15‐min interval. When administered prior to HBO2 exposure, both naltrexone and L‐NAME antagonized the antinociceptive effect seen 90 min later. When administered prior to antinociceptive testing, naltrexone but not L‐NAME antagonized the antinociceptive effect of HBO2. These results suggest that there is a critical role of NO during HBO2 exposure that is required for expression of the opioid‐mediated antinociceptive effect 90 min later. (Supported in part by NIH Grant GM‐77153, ASPET SURF Program and the Chico Hyperbaric Center).

Comparison of the antinociceptive effects of two pharmacological gases, nitrous oxide (N2O) and hyperbaric oxygen (HBO2)

HBO2 treatment is reportedly effective in alleviating various chronic pain conditions (Yildiz et al., Curr. Pain Headache Rep. 10:95–100, 2006). To investigate the mechanism of action, male NIH Swiss mice (20–30 g) were exposed to HBO2 (100% O2 @ 2.5 ATA) and assessed for antinociceptive responsiveness using the glacial acetic acid abdominal constriction test conducted in a small animal hyperbaric chamber. This 10‐min exposure evoked a robust antinociceptive effect during the HBO2 exposure. The magnitude of the HBO2‐induced antinociceptive was significantly reduced by pretreatment with naloxone and L‐NAME and partly antagonized by pretreatment with an antiserum against rat dynorphin. By comparison, the magnitude of antinociceptive effect of 70% N2O was significantly attenuated by pretreatment with naloxone, L‐NAME and dynorphin antiserum. These results indicate that HBO2–induced antinociception has commonalities with N2O. HBO2, similar to N2O, seems to evoke an NO‐dependent, opioid receptor‐mediated antinociceptive effect that may involve a different endogenous opioid peptide. (Supported in part by NIH Grant GM‐77153, ASPET SURF Program and the Chico Hyperbaric Center).

Comparison of the opioid receptor antagonist properties of naltrexone and 6β-naltrexol in morphine-naïve and morphine-dependent mice

It has been proposed that on chronic morphine treatment the µ-opioid receptor becomes constitutively active, and as a consequence, the opioid withdrawal response arises from a reduction in the level of this constitutively active receptor. In support of this, the putative µ-opioid receptor inverse agonist naltrexone has been shown to precipitate more severe withdrawal behavior in mice than the putative neutral receptor antagonist 6β-naltrexol. In the present study naltrexone and 6β-naltrexol were compared in NIH Swiss mice to test the hypothesis that their differential ability to precipitate withdrawal is due to differences in their in vivo opioid receptor antagonist potencies caused by differential access to µ-opioid receptors in the central nervous system and not necessarily by intrinsic differences in their opioid receptor activity. In naïve mice both compounds had similar potencies to antagonize morphine-induced antinociception in the hot plate and warm-water tail-withdrawal assays when measured under equilibrium conditions and afforded similar calculated apparent in vivo µ-opioid receptor affinities. In morphine-dependent mice both compounds precipitated withdrawal jumping but naltrexone was between 10- and 100-fold more potent than 6β-naltrexol. A similar potency difference was seen for other withdrawal behaviors. Both naltrexone and 6β-naltrexol at 1 mg/kg reversed antinociception induced by the long-lasting µ-opioid receptor agonist BU72 in the warm-water tail-withdrawal assay, but antagonism by naltrexone was 6-fold more rapid in onset at equal doses. Since the compounds have similar affinity for the µ-opioid receptor in vivo, the results suggest that the differences observed between the ability of naltrexone and 6β-naltrexol to precipitate withdrawal in the mouse may be explained by differential onset of receptor antagonist action.

Oncogenicity of DNA in vivo: Tumor induction with expression plasmids for activated H- ras and c- myc

All vaccines and other biological products contain contaminating residual DNA derived from the production cell substrate. Whether this residual cell-substrate DNA can induce tumors in vaccine recipients and thus represent a risk factor has been debated for over 50 years without resolution. As a first step in resolving this issue, we have generated expression plasmids for the activated human H- ras oncogene and for the murine c- myc proto-oncogene. Their oncogenic activity was confirmed in vitro using the focus-formation transformation assay. Two strains of adult and newborn immune-competent mice were inoculated with different amounts of either plasmid alone or with a combination of the H- ras and c- myc plasmids. Tumors developed only in mice inoculated with both plasmids and only at the highest amount of DNA (12.5 μg of each plasmid). The NIH Swiss mouse was more sensitive than the C57BL/6 mouse, and newborn animals were more sensitive than adults. Cell lines were established from the tumors. PCR and Southern hybridization analyses demonstrated that both inoculated oncogenes were present in all of the tumor-derived cell lines and that the cells in the tumors were clonal. Western analysis demonstrated that both oncoproteins were expressed in these cell lines. These results demonstrate that cellular oncogenes can induce tumors following subcutaneous inoculation. Such information provides a possible way of evaluating and estimating the theoretical oncogenic risk posed by residual cell-substrate DNA in vaccines.