Tail Injection Research Articles

Folate-PEG-CKK2-DTPA, A Potential Carrier for Lymph-Metastasized Tumor Targeting

A novel conjugate, Folate-PEG-CKK(2)-DTPA, was designed and prepared as a carrier for lymphatic metastasized tumor imaging diagnosis and targeting therapy. Folate-PEG-CKK(2)-DTPA was synthesized and characterized by analysis High Performance Liquid Chromatography, Size Exclusive Chromatography and (1)H-NMR. (99m)Tc-labeled conjugation was prepared, and in vivo quantitative biodistribution and SPECT imaging were studied after subcutaneously injected into the rats and rabbits, respectively. Cell uptake study was carried in a KB cell line using fluorescent methods. In vivo and ex vivo fluorescent imaging study was carried in tumor-bearing nude mouse to evaluate its targeting ability. Folate-PEG-CKK(2)-DTPA was synthesized with high purity. Both in vivo biodistribution study and SPECT imaging study show the rapid direction and high distribution of the conjugation to the lymph nodes. The uptake of fluorescence-labeled Folate-PEG-CKK(2)-DTPA in human oral epidermis carcinoma cells was observed. In vivo and ex vivo fluorescent imaging study indicated it could accumulate in tumor region after vein tail injection in nude mouse. All these findings suggested Folate-PEG-CKK(2)-DTPA as a novel and dependable carrier for tumor diagnosis and therapy, especially for lymph-metastasized tumors.

Investigation of the Anti-Platelet Activity of a Novel Function-Blocking Antibody Targeting the Human Thromboxane A2 Receptor Protein.

Abstract 4019Poster Board III-955Despite the well-documented involvement of thromboxane A2 receptor (TPR) signaling in the pathogenesis of thrombotic diseases, there are currently no rationally designed antagonists available for clinical use. To a large extent this derives from a lack of knowledge regarding the topography of the TPR ligand binding pocket. To this end, while previous work has defined the C-terminal segment of the second extracellular loop (C-EL2) of TPR as the ligand binding site, we have recently mapped the amino acid residues, within this region, which mediate ligand interaction. Since these studies employed a polyclonal antibody targeting the EL2 region of the receptor protein, and since EL2 has been shown to contain the ligand binding domain, we hypothesized that this antibody (referred to hereafter as C-EL2Ab) would exhibit biological activity. On this basis, the purpose of the current study was to investigate the capacity of C-EL2Ab to block TPR-mediated function, using human and mouse platelets. Our initial results demonstrated the C-EL2Ab blocked human and mouse platelet aggregation triggered by 1μM of the TPR agonist U46619, or 0.5mM of the thromboxane A2 mimetic arachidonic acid, in a dose-dependent fashion (150-250nM). On the other hand, control experiments revealed that C-EL2Ab, even at 250 nM, did not produce any detectable effects on platelet activation by ADP (15μM) or the thrombin receptor activating-peptide 4 (40μM). Together, these findings suggest that C-EL2Ab acts as a selective antagonist for TPRs. Consistent with this notion, we found that C-EL2Ab has the ability to displace a radio-labeled TPR antagonist (i.e., [3H]SQ29,548) from its TPR binding sites, both in human and mouse platelets. We next examined the anti-platelet activity of C-EL2Ab under ex vivo experimental settings. It was found that intravenous (IV) tail injections of 250nM of C-EL2Ab resulted in significant blockade of platelet aggregation by 1μM U46619, but not by 15μM ADP. Finally, using a mouse carotid artery thrombosis model, our data demonstrated that C-EL2Ab (at 250 nM; administered IV) prolonged the time for occlusion. Thus, C-EL2Ab has the potential to protect against thrombosis development; which is consistent with the established link for TPRs in thrombotic disorders. In summary, these studies show that C-EL2Ab dose-dependently blocks platelet aggregation induced by the TPR pathway, under both in vitro and ex vivo conditions, and that these effects are mediated via direct interaction with the receptor protein. Furthermore, IV injections of C-EL2Ab are found to confer protection against thrombosis development, in mice. Collectively, these results clearly demonstrate that C-EL2Ab has anti-platelet/anti-thrombotic effects, mediated via its binding to platelet TPRs, making it the first function-blocking antibody against these receptors. Moreover, the identification of a functionally active TPR sequence will significantly aid molecular modeling study predictions for organic derivatives which possess in vivo activity. Disclosures:No relevant conflicts of interest to declare.

Expression and characterization of Momordica Chanrantia anti-hyperglycaemic peptide in Escherichia coli

A nucleic acid sequence MC, encoding Momordica Chanrantia anti-hyperglycaemic peptide MC6 (accession: AAX06814) synthesized according to Escherichia coli preferred codons, was cloned and expressed in E. coli. Recombinant protein pQE8-MC (about 3.5 kDa) was purified and analyzed by 20% SDS-PAGE and western blot. It revealed that the expressed pQE8-MC had good solubility in aqueous media. An HPLC assay was used to confirm the expression of pQE8-MC. Subsequent pharmacological activity assay revealed a significant hypoglycemic effect of low dose treatments of pQE8-MC on male kunming mice. Four hours after an intravenous tail injection, the blood sugar levels of mice treated with pQE8-MC saline solution A3 (1 mg/kg BW) decreased greatly (P < 0.01) relative to the levels of a control group. This suggests that pQE8-MC, expressed in bioengineered E. coli, has a similar hypoglycemic function to the natural protein MC6 from M. Chanrantia. These results reveal the possibility of using bio-engineered bacteria as an anti-diabetic agent.

QX-314 Produces Long-lasting Local Anesthesia Modulated by Transient Receptor Potential Vanilloid Receptors in Mice

The quaternary lidocaine derivative QX-314 is now known to produce long-lasting local anesthesia despite its positive charge. However, recent research suggests that the transient receptor potential vanilloid receptor agonist, capsaicin, should reduce the onset and offset times, whereas the transient receptor potential vanilloid receptor antagonist, capsazepine, should delay the onset time of sensory blockade by QX-314. Sensory blockade in the tail of the conscious mouse was investigated using QX-314 2.5% in combination with capsaicin 0.1% and/or capsazepine (50 microg/ml). After tail injection, onset and offset times of local anesthesia were measured using the hot water tail-flick latency test. Capsaicin reduced the onset time of local anesthesia by QX-314 by more than 75% (Mann-Whitney test, P = 0.007; n = 10 per group) with no effect on the offset time of QX-314. For QX-314 without capsaicin, the onset and offset times were 23 min (interquartile range 15-30 min) and 300 min (interquartile range 285-375 min), respectively. For QX-314 with capsaicin, the onset and offset times were 4 min (interquartile range 3-8 min) and 360 min (interquartile range 285-435 min), respectively. In the antagonist study, capsazepine without added capsaicin decreased QX-314's efficacy, as 6 out of 9 mice did not develop sensory blockade after 90 min (Fisher exact test, P = 0.009). We have confirmed in a sensory blockade model that QX-314 is a local anesthetic with a slow onset and a long duration of reversible blockade. Capsaicin, a transient receptor potential vanilloid receptor agonist, accelerated QX-314's onset kinetics, whereas capsazepine, a transient receptor potential vanilloid receptor antagonist, decreased QX-314's efficacy. These observations raise the possibility that endovanilloids may modulate cell entry of QX-314.

Distribution of 99mTc-dimercaptosuccinic Acid after Intramuscular Injection in the Caudal Limb and Tail of Green Iguanas, Iguana iguana, Using Nuclear Scintigraphy

ABSTRACT Six sub-adult green iguanas, Iguana iguana, were assigned to two groups of three subjects each in a crossover study designed to investigate the renal portal system through the evaluation of tail and hind limb intramuscular (IM) injections. In trial 1, iguanas received an IM injection of 99mTc-dimercaptosuccinic acid (99mTc-DMSA) either in the tail (n = 3) or in the hind limb (n = 3); the iguanas were injected in the other site during trial 2. Nuclear scintigraphy was used to measure isotope gamma particles emitted in counts per minute and to follow distribution of the 99mTc-DMSA postinjection. Dynamic study ventral view scintigrams, with counts per minute measured every 5 sec for the first 5 min after injection and then summed, were used to compare 99mTc-DMSA distribution in the cranial and caudal regions. These revealed that 99mTc-DMSA injected IM into the hind limb remained only in the caudal body for three of the iguanas (50%) and was distributed to both the cranial and caudal regions in the r...

Analgesia Induced by Morphine Microinjected into the Nucleus Raphe Magnus: Effects on Tonic Pain

One of the possible sites of action of the analgesic effect of morphine is the Nucleus Raphe Magnus, as morphine injected into this structure induces analgesia in transient pain models. In order to test if morphine in the Nucleus Raphe Magnus is also analgesic in a tonic pain model, 5 microg of morphine or saline (control) were microinjected into the Nucleus Raphe Magnus of the rat. Analgesic effects were assessed following nociceptive stimulation using transient heating of the tail (phasic pain) and subcutaneous orofacial injection of 1.5 % formalin (tonic pain). While morphine was strongly analgesic for the tail-flick response (p <0.0001 compared to control), analgesia on the response to formalin was also observed for both early (p = 0.007) and late responses (p = 0.02). However, the response to formalin was not completely blunted. These results suggest that the Nucleus Raphe Magnus is not the exclusive site of action of morphine-induced analgesia in clinical conditions.

Identification of Pancreatic Cancer Stem Cells

Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. Although data have been provided to support this theory in human blood, brain, and breast cancers, the identity of pancreatic cancer stem cells has not been determined. Using a xenograft model in which primary human pancreatic adenocarcinomas were grown in immunocompromised mice, we identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD44, CD24, and epithelial-specific antigen (ESA). Pancreatic cancer cells with the CD44(+)CD24(+)ESA(+) phenotype (0.2-0.8% of pancreatic cancer cells) had a 100-fold increased tumorigenic potential compared with nontumorigenic cancer cells, with 50% of animals injected with as few as 100 CD44(+)CD24(+)ESA(+) cells forming tumors that were histologically indistinguishable from the human tumors from which they originated. The enhanced ability of CD44(+)CD24(+)ESA(+) pancreatic cancer cells to form tumors was confirmed in an orthotopic pancreatic tail injection model. The CD44(+)CD24(+)ESA(+) pancreatic cancer cells showed the stem cell properties of self-renewal, the ability to produce differentiated progeny, and increased expression of the developmental signaling molecule sonic hedgehog. Identification of pancreatic cancer stem cells and further elucidation of the signaling pathways that regulate their growth and survival may provide novel therapeutic approaches to treat pancreatic cancer, which is notoriously resistant to standard chemotherapy and radiation.

Interferon-β Fails to Protect in a Model of Transient Focal Stroke

Compelling evidence supporting the role of inflammation in the development of cerebral infarction has focused attention on the potential of antiinflammatory treatment strategies for stroke. Interferon (IFN)-beta, an immunomodulatory agent approved for treatment of multiple sclerosis, is being evaluated in a phase I clinical trial in acute ischemic stroke. In the present study, we evaluated the effects of wild-type rat IFN-beta and its pegylated counterpart (PEG-IFN-beta) in a model of focal ischemia and reperfusion. After 60 minutes of middle cerebral artery occlusion, rats (n=12/group) were treated with IV tail injections of 8 or 16 mug of IFN-beta in 300 muL of PBS once daily for 3 or 7 days or with IV or SC injections of PEG-IFN-beta for 1 day. The animals were assessed daily for weight and for neurological findings. Additional animals underwent complete hematology and chemistry profiles, as well as complete multiorgan necropsy studies. All of the brain tissue was evaluated for assessment of infarct areas, neutrophil infiltration, and presence of hemorrhagic transformations. IFN-beta and PEG-IFN-beta failed to protect against experimental ischemic brain injury as assessed by histopathology and neurological outcome. Furthermore, IFN-beta treatment was associated with significant weight loss and alterations in hematology and chemistry profiles. Our results suggest that additional preclinical studies are warranted.

99mTcOAADT]-(CH2)2-NEt2: A Potential Small-Molecule Single-Photon Emission Computed Tomography Probe for Imaging Metastatic Melanoma

Evaluation of [99mTc]oxotechnetium(V) complexes of the amine-amide-dithiol (AADT) chelates containing tertiary amine substituents as small-molecule probes for the diagnostic imaging of metastatic melanoma has shown that technetium-99m-labeled AADT-(CH2)2-NEt2 (99mTc-1) has the highest tumor uptake and other favorable biological properties. We have, therefore, assessed this agent in a more realistic metastatic melanoma model in which, after i.v. tail injection, a highly invasive melanoma cell line, B16F10, forms pulmonary tumor nodules in normal C57BL6 mice. Small melanotic lesions develop in the lungs and, on histologic examination, appear as small black melanoma colonies, increasing in size and number with time after tumor cell injection. Groups of mice received tumor cell inocula of 2 x 10(5), 4 x 10(5), or 8 x 10(5) B16F10 cells; 14 days later, 2 hours after 99mTc-1 administration, lung uptake of 2.83 +/- 0.21%, 3.63 +/- 1.07%, and 4.92 +/- 1.61% injected dose per gram of tissue (% ID/g), respectively, was observed, compared with normal lung uptake of 2.13 +/- 0.2% ID/g (P < 0.05). Additionally, a higher level of 99mTc-1 accumulation was seen 17 days after tumor cell inoculation as the lung lesions grew. These in vivo studies coupled with additional in vitro and ex vivo assessment show that 99mTc-1 has high and specific uptake in melanoma metastases in lungs and can potentially follow the temporal growth of these tumors.

Synthetic tubulin inhibitor HTI-286 and hepatocellular carcinoma: an in-vitro and and in-vivo study

4201 Background: HTI-286 (HTI) is a synthetic tubulin inhibitor and a hemiasterlin analogue. It is a potent inhibitor of cell growth with an additional advantage of circumventing the P-glycoprotein-mediated resistance, which hampers efficacy of several antimicrotubule agents. Aim of our study was to investigate in-vitro antiproliferative effect of HTI in several hepatic cancer cell lines (Morris hepatoma (MH), Hep G2 and Hep 3B) and on primary human hepatocytes. Furthermore tumor growth inhibition was analyzed in a rat in-vivo study. Methods: MTT assay was done to determine the doubling time of each cell line. Exponentially growing cells were seeded with different concentrations of HTI for several doubling times. We determined the cytotoxicity and IC(50) by the MTT method. Microtubule structure was visualized by alpha-tubulin-immunofluoresence (aTIF). All animal experiments were approved by the local ethic commission. MH were implanted into American cancer institute rats (ACI) and tumor growth was pursued with MR-imaging (MR). HTI was administered via tail injection at several time points. The tumor growth was followed for over a period of one month. Results: In MH, HepG2 and Hep3B cells HTI has shown an antiproliferative effect already after one doubling time (22, 48 and 55 hours). IC(50) in MH cells was around 1 nM. Cytotoxic effect of HTI rose with increasing concentration. Proliferation of primary human hepatocytes was not affected by the HTI-treatment. aTIF showed a significant difference in HTI-treated compared to non-treated cells. The in-vivo study revealed significant growth inhibition in HTI treated group compared to a saline solution treated group. Conclusions: Our in-vitro data show that HTI inhibits proliferation of hepatic tumor cells in-vitro and tumor growth in-vivo. aTIF confirmed disrupture of microtubule organization. Furthermore primary human hepatocytes do not seem to be affected by the HTI treatment. The data suggest HTI to be considered as a potent promising cytotoxic and antiproliferative drug in liver oncology especially as it circumvents the P-glycoprotein-mediated drug resistance, being responsible for resistance to taxanes and vinca alkaloids. No significant financial relationships to disclose.

W14-P-004 A thrombomodulin haplotype (V/DELTT),associated with risk of coronary heart disease, does not influence plasma levels of soluble thrombomodulin

Neuronal damage after transient cerebral ischemia is exacerbated by signaling pathways involving activated platelet-activating factor (PAF) and ameliorated by PAF-acetylhydrolase (PAF-AH); but whether cerebral neurons can be rescued by human recombinant PAF-AH (rPAF-AH) remains unknown. Adult male mice underwent a 60 min middle cerebral artery occlusion (MCAO) and reperfusion for 24 h. Then, the mice received intravenous tail injections with different drugs. Neurological behavioral function was evaluated by Bederson's test, and cerebral infarction volume was assessed with tetrazolium chloride (TTC) staining. mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2, collagenase-1), MMP-9 (gelatinase-B), and vascular endothelial growth factor (VEGF) were determined by quantitative real-time PCR (RT-PCR) and western blot analysis, respectively. Compared with the vehicle group, rPAF-AH significantly improved sensorimotor function (42%, P = 0.0001). The volume of non-infarcted brain tissue was increased by the rPAF-AH treatment (16.3 ± 4.6% vs. 46.0 ± 10.3%, respectively). rPAF-AH significantly reduced mRNA and protein levels of MMP-2 and MMP-9, but increased the mRNA (P < 0.001) and protein levels (P < 0.01) of VEGF. These results demonstrate that rPAF-AH provides neuroprotection against ischemic injury. Neuroprotection might be induced not only by decrease in MMP-2 and MMP-9 expression, but also by increased VEGF expression.

Relaxatory effect of magnesium on mesenteric vascular beds differs from normal and streptozotocin induced diabetic rats

Magnesium deficiency has recently been proposed as a novel factor implicated in the pathogenesis of diabetes complications. Previous studies have shown that magnesium decreases basal tone in normal isolated aortic rings and reduces phenylephrine-induced contraction. The mechanism of this magnesium action is not very well known. The present study was designed to determine the role of endothelium and nitric oxide in magnesium sulfate-induced vasorelaxation in diabetic rat vessels. Diabetes was induced by a single tail injection of streptozotocin. Eight weeks later, superior mesenteric arteries of control and diabetic animals were isolated and perfused according to the McGregor method. Prepared vascular beds were constricted with phenylephrine to induce 70–75% of maximal constriction. Magnesium sulfate at concentrations of 0.001 M to 0.1 M was added into the medium and perfusion pressure was then recorded. Mesenteric bed baseline perfusion pressure in intact and denuded endothelium of diabetic groups was higher than controls. In all groups, relaxant response to magnesium in mesenteric bed was attenuated after endothelium removal, but a relaxatory effect appears at high concentration. In the presence of N (ω)-nitro- l-arginine methyl ester ( l-NAME), magnesium-induced relaxation was significantly suppressed in intact mesenteric bed of control animals but in diabetics, the relaxant response was slightly inhibited. From the results of this study, it can be concluded that magnesium-induced endothelium dependent and endothelium independent vasorelaxation are mediated by nitric oxide in control rats while in diabetic animals other mechanisms may be involved.

ACUTE NICOTINE TREATMENT ACCELERATES PHOTOCHEMICALLY INDUCED PLATELET AGGREGATION IN CEREBRAL ARTERIOLES OF MICE: AN IN VIVO STUDY

When tobacco is smoked, chewed or snuffed, nicotine is absorbed by the lungs or mucous membrane and quickly moved into the bloodstream, where it is circulated throughout the brain. In fact nicotine is highly dangerous to be consumed in any form. The present study was conducted to know the adverse effects of nicotine on the platelet aggregation in cerebral microvessels of mice. Male mice of average weight 33 g were injected with Saline (control) or Nicotine (1 mg/kg, 0.1 ml/10 g) one hour before the experiment. Animals were anaesthetized and trachea was intubated. Craniotomy was performed and a window was opened on the skull. Layer of dura was removed. Brain surface microvessels were exposed and animal was placed on the microscope stage. Microscope was connected to a monitor and VCR to record all events. Exposed brain surface was continuously irrigated with ACSF solution. After the body temp was maintained at 37? C, Sodium fluorescein (2%, 0.1 ml/10 g) was injected i.v. through tail injection. High intensity mercury light was switched on to induce photochemically induced platelet aggregation. Appearance of the 1st platelet aggregation and total blood flow stop were timed in seconds. Our results showed that in the animals treated with nicotine, venule did not show any alteration in the platelet aggregation timings in comparison to the controls. But in arterioles platelet aggregation timings were significantly accelerated (P

Inflammation: Proteinase-Activated Receptor-1 is involved in modulation of acute and chronic inflammatory hyperalgesia

Proteinase-Activated Receptors (PARs) are G-protein-coupled receptors that are activated by proteinases. PAR-1, a member of this family is activated by the proteolytic cleavage of its N-terminal domain by thrombin. Because of functional PAR-1 presence on primary sensory neurons and because we have shown that PAR-1 agonists attenuated nociception, we studied the role of PAR-1 in modulating nociception in physiological or inflammatory conditions. Wild-type (PAR-1+/+) and PAR-1-deficient (PAR-1-/-) mice received an intraplantar injection of formalin (10 μl of 5% formalin) known to cause acute hyperalgesia, or tail injections with collagen type II (0.05 mg/mouse) to induce arthritis. Inflammatory edema was followed for all groups by measuring paw diameter. Nociception in response to thermal or mechanical stimuli was assessed using plantar test apparatus or von Frey filaments respectively, before and after inducing inflammation. Nociceptive scores in response to 3 von Frey filaments of different sizes or withdrawal latencies in response to 3 different thermal intensities were not different in PAR-1+/+ versus PAR-1-/- mice in basal conditions. Formalin intraplantar injection caused significant edema from 15-min. to 7-hours after its injection, similarly in PAR-1+/+ and PAR-1-/- mice. Nociceptive responses to mechanical stimuli showed that formalin intraplantar injection caused allodynia and hyperalgesia in all groups. However, PAR-1-/- exhibits significantly higher nociceptive scores, more allodynia and increased thermal hyperalgesia compared to PAR-1+/+ mice. Similarly, allodynia and hyperalgesia observed in groups of mice after the induction of arthritis was significantly higher in PAR-1-/- mice compared to PAR-1+/+ mice. These results show that PAR-1-/- mice exhibit similar physiological nociception as PAR-1+/+. However, in acute or chronic inflammatory conditions, PAR-1-/- mice experienced more allodynia and hyperalgesia than wild type, this effect not being related to the inflammatory response. These results suggest that PAR-1 activation plays an active role in endogenous inhibition of inflammatory hyperalgesia and allodynia.

Immobilization of North American porcupines (Erethizon dorsatum) using ketamine and xylazine.

We performed 345 immobilizations on 150 North American porcupines (Erethizon dorsatum) using a mixture of ketamine hydrochloride (KH) and xylazine hydrochloride (XH). A subsample of 184 immobilizations performed on 124 individuals from 4 May to 7 November 2000 and from 22 January to 30 April 2001 is thoroughly analyzed. In contrast to published procedures, we found that injecting drugs into tail muscles was more efficient than into longitudinal muscles of the lower back, because tail injections decreased the need of multiple injections by 26%. Using tail injections, we were able to reduce the dose by 50% from other published reports without significantly affecting induction, immobilization, standing, or recovery times. We suggest that injection of 5 mg KH/kg and 2 mg XH/kg in the tail as a standard procedure to immobilize North American porcupines. Body mass significantly affected the induction and standing times for single injections performed in the tail, irrespective of dose or sex. Sex, dose, and mass had no effect on the quality of immobilizations and the respiration rate of individuals during immobilization. We report a 0.87% mortality rate using a mixture of KH and XH and suggest ways to further decrease this rate.

Systemic pre-treatment with a group II mGlu agonist, LY379268, reduces hyperalgesia in vivo.

1. Previous studies investigating the role of metabotropic glutamate (mGlu) receptors in nociceptive processing have been hampered by the lack of systemically active, selective, ligands. This study investigates the possible analgesic and/or anti-hyperalgesic properties of the most potent compound to date that has systemic agonist activity at group II mGlu receptors, LY379268. 2. In testing the drug in rats as an analgesic to acute noxious stimuli, LY379268 (in doses up to 3 mg kg(-1) i.p.) did not affect withdrawal latencies to either mechanical or thermal stimulation. 3. However, when a 3 mg kg(-1) dose was given prior to an intraplantar injection of carrageenan, the inflammatory hyperalgesia that developed was significantly delayed compared to saline pre-treated controls, without affecting the inflammation of the paw. A similar dose of the mGlu-inactive enantiomer, LY379267, was not anti-hyperalgesic. 4. In a model of mouse tail withdrawal to warm water, LY379268 (12 mg kg(-1) i.p.), given before a subcutaneous tail injection of capsaicin, reduced the subsequent neurogenic hyperalgesia. 5. Rota-rod testing showed that the drug did not produce a motor impairment in rats at antihyperalgesic doses. 6. The results indicate that systemic activation of this group of mGlu receptors reduces both inflammatory and neurogenic thermal hyperalgesia.

The synergistic interaction between midazolam and clonidine in spinally-mediated analgesia in two different pain models of rats.

Both midazolam, a benzodiazepine gamma-aminobutyric acid type A receptor agonist, and clonidine, an alpha2-adrenergic receptor agonist, induce spinally-mediated analgesia. We investigated the analgesic interaction of spinally-administered midazolam and clonidine in their effects on acute and inflammatory nociception. Rats implanted with lumbar intrathecal catheters were injected intrathecally with saline (control), midazolam (1 to 100 microg), or clonidine (0.1 to 3 microg) to test for their responses to thermal stimulation to the tail (tail-flick test) and subcutaneous formalin injection into the hind paw (formalin test). The effects of the combination of midazolam and clonidine on both stimuli were tested by isobolographic analysis by using the 50% effective doses. The general behavior and motor function were examined as side effects. When combined, the 50% effective doses of midazolam (clonidine) decreased from 1.57 microg (0.26 microg) to 0.29 g (0.05 microg) in the tail-flick test and from 1.34 microg (0.12 microg) and 1.21 microg (0.13 microg) to 0.05 microg (0.005 microg) and 0.13 microg (0.015 microg) in Phase 1 and 2 of the formalin test, respectively. Side effects did not increase by using the combination. These results suggest a favorable combination of intrathecal midazolam and clonidine in the management of acute and inflammatory pain after proper neurotoxicologic studies. Spinally-administered midazolam, a benzodiazepine, and clonidine, an alpha2-adrenergic receptor agonist, have significant synergistic effects on thermally-induced acute and formalin-induced inflammatory pain.

Long-Lasting Regulation of Galanin, Opioid, and Other Peptides in Dorsal Root Ganglia and Spinal Cord during Experimental Polyarthritis

Mechanisms involved in transition from acute to chronic pain are still not well understood and our means to therapeutically influence this transition are limited. Moreover, very little is known about long-lasting consequences of prolonged exposure to painful stimuli with regard to phenotypic changes and pain experience. In this study we have analyzed long term behavioral and neurochemical effects of intradermal tail injection of heat-killed mycobacterium butyricum suspended in complete Freund's adjuvant. Calcitonin gene-related peptide (CGRP) and galanin mRNA levels were investigated in dorsal root ganglia of polyarthritic rats during the acute (21-) and the remission stage (79 days postinjection), and opioid peptide mRNAs and receptors were studied in the spinal cord. Most of the increases in peptide mRNA levels observed during the acute stage of arthritis were still present in the remission stages. Thus, CGRP and galanin mRNAs in DRGs, and opioid peptide mRNAs and opioid receptors in the spinal cord were still strongly up-regulated, when animals do not exhibit spontaneous pain behavior and inflammation. Hot-plate test in the presence of naloxone, performed in the remission stage, indicated that opiates participate in pain threshold regulation after prolonged painful condition. Finally, X-ray examination revealed a complete destruction of joint structure, thus suggesting a parallel lesion of peripheral nerve endings. These results suggest that in the remission stage of chronic joint inflammation several types of mechanisms are activated aiming at counteracting both inflammatory and neuropathic pain. Thus, opioid systems in the dorsal horn as well as galanin in DRG neurons are upregulated, both alternating pain.

Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic Bcl-2 gene.

To examine the possibility of reducing ischemia-reperfusion injury (I/R injury) to the mouse liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic human Bcl-2 gene. Ischemia-reperfusion injury has been demonstrated in a number of clinically relevant diseases such as myocardial infarction, cerebrovascular disease, sepsis, peripheral vascular disease, and organ transplantation. In this regard, apoptosis plays a central role. Normal C57BL/6 mice were used. An adenovirus (deltaE1) vector containing the human Bcl-2 gene was developed in the authors' laboratory. An adenovirus vector encoding an irrelevant gene (beta-galactosidase, AdCMVLacZ) was used as a control. Taking advantage of the hepatotropic properties of adenovirus vectors, gene transfer was performed with 1 x 10(9) plaque-forming units by intravenous tail injection, 48 hours before the ischemic injury. Ischemic-reperfusion injury was induced by temporal and segmental occlusion of hepatic blood flow. Aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase activity was measured using standard assays. Liver biopsies were obtained before and 6 hours after I/R injury for morphologic assessment, and apoptosis was determined in situ with a histochemical assay. The expression of AdCMVhBcl-2 vector was confirmed by reverse transcription-polymerase chain reaction and functionally validated in apoptotic studies in endothelial cells. Expression of the Bcl-2 gene protects against I/R injury, as shown by a significant decrease in transaminases (p < 0.05) and necrosis and apoptosis (p < 0.001), and permanent survival (p < 0.0001), compared with sham-operated animals and animals treated with AdCMVLacZ. Genetic modification of the liver to induce cytoprotection has potential applications to prevent I/R injury to the liver in surgical interventions, including liver transplantation.

高周波熱プラズマで処理したチタンカーバイド粉末のAl&lt;SUB&gt;2&lt;/SUB&gt;O&lt;SUB&gt;3&lt;/SUB&gt;-TiC複合セラミックスヘの応用

In order to investigate the influence of plasma treatment on the powder properties, titanium carbide powder with an average particle size of 1.35 μm was modified by the in-flight induction plasma treatment generated at the plate power of 45 kW and reactor pressure of 26.7 kPa. The treatment in Ar-H2 induced the formation of carbon-site vacancies. The tail injection of Ar-NH3 to the Ar-H2 plasma resulted in the partial substitution of carbon by nitrogen. Also, the treatment in Ar-N2 plasma gave rise to the partial nitridation. A large amount of oxygen incorporated into the plasma-treated powders stored in air. The original and plasma-treated powders were dispersed in an alumina matrix, and the pressureless sintering of Al2O3-TiC composites was tried to elucidate the influence of plasma modification. The composites with the dispersion of plasma-treated titanium carbide powders showed an increase in sintered density.