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Summary We have developed an ultrasound probe through the centre of which an epidural needle can pass, intended to reduce the rate of contact between bone and needle during epidural insertion. We tested the ability of this probe to identify the lumbar interspace, using A-mode ultrasound, in a submerged plastic model, a porcine phantom and five human volunteers. In the plastic model, the minimum echo representing the interspace was only 8.8% of the maximum echo from the ‘bone’. In the porcine model, the echo variations between the interspace and L3 were up to 48% and the needle was safely inserted into the interspace without bone contact under guidance. The human study also showed that the maximum bone echo was at least three times stronger than the interspace echo. Axial ultrasound guidance, with the needle passing through the probe, offers a method for reducing bone contact during epidural insertion.

Background Nerve conduction block using high-intensity focused ultrasound (HIFU) has been conducted with nerves of mixed fibres in normal animal models. This study tested the feasibility and safety of HIFU for sensory nerve conduction block in diabetic neuropathic nerves to determine its potential for pain relief. Methods Diabetes was induced in Sprague–Dawley rats using streptozotocin, and HIFU at 2.68 MHz was used for the block. This study consisted of two sections, in vitro and in vivo. For the in vitro experiments, the entire contiguous sciatic–sural nerves were obtained. Compound action potentials and sensory action potentials were recorded in the sciatic and sural nerves, respectively. For the in vivo experiments, compound muscle action potentials (CMAPs) were recorded from the gastrocnemius muscles. All data were expressed as median (range). Results The in vitro results showed that HIFU temporarily inhibited sensory action potentials of the control and diabetic rat nerves to 33.9 (8.2) and 14.0 (10.7)% of the baseline values, respectively, whereas the compound action potentials were suppressed to 53.6 (8.4) and 76.2 (7.5)% of baseline, respectively. The in vivo results showed that HIFU acutely blocked CMAPs to 32.9 (12.6) and 19.9 (10.9)% of baseline in control and diabetic rat nerves, respectively. Measurements of CMAPs and histological exanmination were used for indirect assessment of the safety of the HIFU technique. Conclusions High-intensity focused ultrasound safely and reversibly suppressed nerve conduction in diabetic rat nerves when the stimulation parameters were appropriate. The results suggest that HIFU may have potential to block sensory nerves reversibly and provide peripheral pain relief.

The present study was designed to investigate whether the Electroacupuncture (EA) is beneficial to extenuate cerebral injuries following transient Middle Cerebral Artery Occlusion (MCAO), as well as to observe the effect of EA on expression of Basic Fibroblast Growth Factor (bFGF) -like Immunoreactivity (IR) in rat brains. The results indicate that gross neuronal damages include infarction, swelling and neuron loss, accompanied by increased bFGF-like IR expression following MCAO. In peri-infarct striatum, bFGF-like IR was mainly located in astrocytes except some neurons also showed an upregulation of the IR; in frontoparietal cortex, strong induction of bFGF-like IR was mostly seen in neurons. Both the EA applied during ischemia and reperfusion could evidently alleviate cerebral lesion extent, notably upregulate the expression of bFGF-like IR in striatum and cortex, but there was no significant difference between the effects of EA applied during ischemia and reperfusion, except EA applied during reperfusion seems to be more effective in reducing the cerebral swelling. The results implied that, in striatum, astrocytes might play an important role in the protection of neuron via the expression of bFGF; whereas in cortex, neurons may exert autoprotection through secreting bFGF themselves. One possible protective effect of EA lies in regulating the endogenous expression of bFGF.

The effect of the antifungal drug bifonazole on Ca2+ homeostasis in Madin Darby canine kidney (MDCK) cells was investigated. Cell suspensions were loaded with the Ca2+-sensitive dye fura-2, and the fluorescence changes were measured with a spectrofluorophotometer. At concentrations between 10-80 microM bifonazole increased cytosolic free Ca2+ levels ([Ca2+]i) in a concentration-dependent manner. The Ca2+ signals were partly inhibited by removing extracellular Ca2+. Bifonazole (40 microM) released Ca2+ from the store sensitive to 1 microM thapsigargin, an endopolasmic reticulum Ca2+ pump inhibitor. Bifonazole (40 microM) per se induced capacitative Ca2+ entry while reduced 1 microM thapsigargin-induced capacitative Ca2+ entry. Inositol 1,4,5-trisphosphate may be involved in bifonazole-induced Ca2+ release because inhibiting phospholipase C with 2 microM U73122 partly reduced the bifonazole response. Together, bifonazole increased [Ca2+]i in renal tubular cells by inducing intracellular Ca2+ release and extracellular Ca2+ influx.

The effect of oleamide, a sleep-inducing endogenous lipid in animal models, on intracellular free levels of Ca(2+) ([Ca(2+)](i)) in non-excitable and excitable cells was examined by using fura-2 as a fluorescent dye. [Ca(2+)](i) in pheochromocytoma cells, renal tubular cells, osteoblast-like cells, and bladder cancer cells were increased on stimulation of 50 microM oleamide. The response in human bladder cancer cells (T24) was the greatest and was further explored. Oleamide (10-100 microM) increased [Ca(2+)](i) in a concentration-dependent fashion with an EC(50) of 50 microM. The [Ca(2+)](i) signal comprised an initial rise and a sustained plateau and was reduced by removing extracellular Ca(2+) by 85 +/- 5%. After pre-treatment with 10-100 microM oleamide in Ca(2+)-free medium, addition of 3 mM Ca(2+) increased [Ca(2+)](i) in a manner dependent on the concentration of oleamide. The [Ca(2+)](i) increase induced by 50 microM oleamide was reduced by 100 microM La(3+) by 40%, but was not altered by 10 microM nifedipine, 10 microM verapamil, and 50 microM Ni(2+). In Ca(2+)-free medium, pre-treatment with thapsigargin (1 microM), an endoplasmic reticulum Ca(2+) pump inhibitor, abolished 50 microM oleamide-induced [Ca(2+)](i) increases; conversely, pretreatment with 50 microM oleamide reduced 1 microM thapsigargin-induced [Ca(2+)](i) increases by 50 +/- 3%. Suppression of the activity of phospholipase C with 2 microM U73122 failed to alter 50 microM oleamide-induced Ca(2+) release. Linoleamide (10-100 microM), another sleep-inducing lipid with a structure similar to that of oleamide, also induced an increase in [Ca(2+)](i). Together, it was shown that oleamide induced significant [Ca(2+)](i) increases in cells by a phospholipase C-independent release of Ca(2+) from thapsigargin-sensitive stores and by inducing Ca(2+) entry.

Cathepsin E is found mainly over the gastric surface and foveolar epithelial cells, and it also is found in the metaplastic pyloric glands and cancer cells. The exact function of cathepsin E in gastric mucosa remains unclear. The colonic type (type III) of intestinal metaplasia (IM) is strongly associated with intestinal-type gastric carcinoma. IM is considered to be a precancerous lesion. The aim of this study was to find out the role of cathepsin E in IM, dysplasia and cancer of stomach.Sixty nine biopsy specimens with IM and dysplasia and 33 gastrectomy specimens with gastric carcinoma were fixed, sectioned and stained with PAS-alcian blue stain, high iron-diamine alcian blue stain to classify IM and immunohistochemical stain to localize cathepsin E. Those patients with dysplastic gastric lesions received regular endoscopic follow-up.Fifteen of 69 patients with gastric dysplasia developed cancer in a median 10.5 months follow-up. Severe dysplasia developed carcinoma significantly higher than mild dysplasia (12/20 vs. 1/25, p0.001), and type III intestinal metaplasia seemed to have significantly predilection for severe dysplasia and gastric cancer. Cathepsin E was stained in intestinal metaplasia with dysplastic change in 44/69 specimens (63.8%), and carcinoma in 28/48 (58.3%) specimens, there was no significant difference between intestinal type and diffuse type carcinoma in cathepsin E staining. The positive staining for cathepsin E decreased significantly in severe dysplastic gastric mucosa.Type III IM is commonly associated with severe dysplasia and cancer; it may be a precancerous lesion. The positive staining of cathepsin E decreased with the severity of gastric dysplasia, representing dedifferentiation of the cells.

In order to investigate the effect of 7-nitroindazole (7-NI) on penicillin-induced epilepsy and the relationship between NO and anti-epileptic effect of electro-acupuncture, computerized physiological polygraph and NO-sensitive electrode with potentiostat were respectively used to record the total power spectrum (TPS) of EEG and the concentration of NO in rat hippocampus pretreated with electro-acupuncture or 7-NI for 30 min before penicillin. The results showed that the TPS of EEG and concentration of NO in hippocampus increased greatly after the epilepsy induction. Treatment with electroacupuncture inhibited the TPS of EEG sharply (P0.01). The latency and threshold of epilepsy induction were decreased by 7-NI, but paroxysm abated and the TPS was inhibited (P0.05) as compared with the control group. Both pretreatments with 7-NI and electroacupuncture decreased the NO concentration (P0.01). The above results suggest that the propagation of penicillin-induced seizures is facilitated by nNOS, but with some increase in the latency and threshold of induction. Electroacupunture may inhibit seizure through decreasing nNOS transcription in hippocampus.

The effect of fendiline, an anti-anginal drug, on cytosolic free Ca2+ levels ([Ca2+]i) in A10 smooth muscle cells was explored by using fura-2 as a Ca2+ indicator. Fendiline at concentrations between 10-50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 of 20 microM. External Ca2+ removal reduced the Ca2+ signal by 75%. Addition of 3 mM Ca2+ increased [Ca2+]i in cells pretreated with fendiline in Ca2+-free medium. The 50 microM fendiline-induced [Ca2+]i increase in Ca2+-containing medium was inhibited by 10 microM of La3+, nifedipine, or verapamil. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ store partly inhibited 50 microM fendiline-induced Ca2+ release; whereas pretreatment with 50 microM fendiline abolished 1 microM thapsigargin-induced Ca2+ release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 50 microM fendiline-induced Ca2+ release. Incubation with 50 microM fendiline for 10-30 min decreased cell viability by 10-20%. Together, the findings indicate that in smooth muscle cells fendiline induced [Ca2+]i increases. Fendiline acted by activating Ca2+ influx via L-type Ca2+ channels, and by releasing internal Ca2+ in a phospholipase C-independent manner. Prolonged exposure of cells to fendiline induced cell death.

Using a nitric oxide (NO)-sensitive platinum microelectrode (SNM) modified with chitosan nickel (II) complex and Nafion (Nafion-CTS (Ni)-Pt), we observed the effect of nitric oxide synthase (NOS) inhibitor 7-nitro-indazole (7-NI) and N omega-nitro-L-arginine (L-NNA) on penicillin-treated hippocampal slices by simultaneously recording the stimulus-evoked field potentials and nitric oxide release from CA1 pyramidal neurons. Our results show: (1) The linear relationship range and detection limit of SNM were 4.5 x 10(-4)-1.0 x 10(-8) mol/L and 5.0 x 10(-8) mol/L respectively. (2) Penicillin (PEN) could elevate NO release, the number and amplitude of stimulus-evoked field potentials spike (SEPS) in concentration-dependent manner; (3) Both 7-NI and L-NNA depressed NO release and partly reversed the effect of penicillin. The above findings suggest that convulsant provoking effect of NO could be inhibited by NOS inhibitors. The NO-sensitive electrode may provide an useful tool for continuous detection of NO in biological tissues.

The time course of alteration in neuronal nitric oxide synthase (nNOS) and the influence of glutamate receptor antagonists on immunoreactivity of nNOS were investigated immunohistochemically in rat hippocampus during penicillin-induced epilepsy. It was found that nNOS-like immunoreactivity in hippocampus increased at 4 h after initiation of seizure and reached a peak at 24 h. When MK-801 (6 micrograms) and DNQX (4 micrograms) were injected respectively into hippocampus 20 min before administration of penicillin, seizure was reduced in strength. Meanwhile, the nNOS-like immunoreactivity in hippocampus was decreased, compared with the group which were administered penicillin alone. These results suggest that the increase of nitric oxide may be related to the injury of neurons during and after epilepsy; the changes' of nNOS may be related to the activity of NMDA and non-NMDA receptors. Neuron protective effect of MK-801 and DNQX may be in part mediated by nNOS.

To investigate the distribution and expression changes of basic fibroblast growth factor (bFGF)-like immunoreactivity (IR) in focal cerebral ischemic rat brain.The expression of bFGF was observed by fluoro-immunohistochemistry. The focal cerebral ischemic injury was carried out using middle cerebral artery occlusion (MCAO) model.Confocal images demonstrated that both the striatum and the frontoparietal cortex showed increases of bFGF-like IR after 2-h MCAO and 24-h reperfusion. In striatum, the increase patterns of bFGF-like IR were different according to the ischemic extent. In the core of infarct and its surrounding region (with more IR induction), bFGF-like IR was mainly located in astrocytes. In region adjacent to infarct (with most IR induction, the grey ratio of bFGF-like IR increased from sham-98% +/- 10% to ischemia-125% +/- 6%), some neurons also showed an upregulation of bFGF-like IR. In frontoparietal cortex, strong induction of bFGF-like IR was mostly seen in neurons (The grey ratio of bFGF-like IR increased from sham-104% +/- 11% to ischemia-132% +/- 28%), although it was observed in astrocytes as well.The expression of bFGF increased after focal cerebral ischemia in rats, suggesting that: in striatum, astrocytes may play an important role in the protection of neurons via the overexpression of bFGF; whereas in cortex, neurons probably exert an autoprotection through expressing bFGF themselves.

In this study, the fluorescent dye Fluo-3 (AM) was applied to rat hippocampus by microinjection, to label neurons in situ. A large number of CA1 pyramidal cells could be labeled by a single injection. With this improvement of labeling and the use of the confocal laser scanning microscope (Leica Lasetechnik GmbH), changes of calcium fluorescence intensity in CA1 pyramidal cells of rat hippocampal slices on the models of mimic epilepsy (induced by penicillin or glutamate) or hypoglycemia/hypoxia were studied in vitro. The results showed that in magnesium free artificial CSF, the intracellular calcium level of the CA1 pyramidal cells increased following application of glutamate or penicillin. Similar phenomenon was also observed on the in vitro model to hypoglycemia/hypoxia.

To explore the role of poly (ADP-ribose) polymerase (PARP) in focal cerebral ischemia with reperfusion injury.Male Wistar rats underwent 3.5-h of temporary middle cerebral artery occlusion by intraluminal suture. Infarction volume was showed with 2,3,5-triphenyltetrazolium chloride (TTC) staining and quantitated by image analysis system, neurologic scores were determined with a 0-5 grading scale.3-Aminobenzamide (3-AB) 10 mg.kg-1 or nicotinamide (Nic) at 20 mg.kg-1 showed potent neuroprotective effects within 0-6 h, neurologic deficits were attenuated. With the increasing dose of PARP inhibitors, beneficial effects were compromised, particularly, administration of Nic 60 mg.kg-1 at the onset of reperfusion drastically accelerated brain damage. Phytomenadione, a selective inhibitor of mono (ADP-ribosyl) transferase, had little effect on infarction volume.Transient incomplete inhibition of PARP provides a neuroprotective effects against cerebral ischemia-reperfusion injury, with a relatively wide therapeutic window, whereas severe inhibition of this enzyme, especially in reperfusion phase, is detrimental.

The present study investigated the role of nitric oxide (NO) in epileptogenesis and whether this role correlated with ionotropic glutamate receptor (IGR). Using a self-constructed NO-sensitive microelectrode (SNM), we observed the effect of nitric oxide synthase (NOS) inhibitors, NMDA and non-NMDA selective antagonists on penicillin(PEN)-treated hippocampal slices by simultaneously recording evoked field potentials and nitric oxide release from CA1 pyramidal neurons. 7-nitroindazole (7-NI),Nomega-nitro-L-arginine (L-NNA) and DL-2-amino-phospho-novaleric acid (APV), but not 6,7-dinitroquinoxaline-2,3 (1h,4h)-dione(DNQX), depressed NO release and partly reversed PEN's epileptogenetic effect, while APV + 7-NI + L-NNA did not display a further inhibitory effect. These findings suggest both NOS inhibitor and NMDA antagonist involve as anticonvulsant factors in epileptogenesis, providing direct evidence for NO release in response to NMDA receptor activation. The anticonvulsant effect of NMDA antagonist may ascribe to its action on NO release.

To study the effect of dextromethorphan (DM) in focal cerebral ischemia.The c-fos protein was detected immunohistochemically in the brain of rats after focal cerebral ischemia (induced by placing a nylon thread in the lumen of the internal carotid artery) with and without treatment with DM.Focal cerebral ischemia induced c-fos protein expression outside the core territory of the middle cerebral artery (MCA) and neuronal damage in the core territory of the MCA. There was an evident expression of c-fos protein in the ipsilateral regions outside the MCA territory (e.g. cingulate cortices, piriform cortices and entorhinal cortices), and in the contralateral regions of hippocampus after 4-h reperfusion following 1-h MCA occlusion. But morphological results showed severe edema and neuronal damage in the core territory and the ipsilateral hippocampus. DM blocked both the c-fos protein induction and neuronal damage in all regions.DM reduced c-fos protein expression and blocked the neuronal damage after focal cerebral ischemia.

To study the neuroprotective effects of dextromethorphan against transient cerebral ischemia/reperfusion injury.Transient cerebral ischemia/reperfusion injury in gerbils was produced by temporarily clamping the common carotid arteries (CCA) for 10 min.Intrahippocampal (ih) injection of 2.0 microL dextromethorphan (DM, 100 mumol.L-1) 5 min before ischemia quickened the recovery of EEG changes, the total power spectra of EEG, and the power of dominant frequency following reperfusion. The total power of EEG was increased to 92 +/- 30 (P0.01) at 240 min following reperfusion. DM substantially reduced the severe ischemic neuronal damage (SIND) after 10 min of cerebral ischemia and 24 h of reperfusion.DM has neuroprotective effects against transient cerebral ischemia and reperfusion injury in gerbils.

Immunocytochemical technics were used to evaluate the influence of penicillin-induced seizure and electroacupuncture treatment on dynorphin1-8 and leu-enkephalin immunoreactivity in hippocampus. It was found that 3 h after beginning of seizure there started a dramatic decrease in dynorphin1-8 in hilus, mossy fiber of hippocampus but an increase in hilus, mossy fiber of hippocampus but an increase in leu-enkephalin in subiculum, CA1 area of hippocampus and some other limbic structures. Electroacupuncture treatment decreased the leu-enkephalin immunoreactivity in the nuclei mentioned above and increased dynorphin1-8 immunoreactivity in hippocampus. The results show that epileptiform activity and electroacupuncture inhibitory effect on seizure may be related to the alteration of dynorphin1-8 and leu-enkephalin in the brain.