In vivo echo-planar imaging of rat spinal cord

An integrated approach to echo-planar imaging of rat spinal cord in vivo with a small field of view (FOV) is presented. This protocol is based on a multishot interleaved echo-planar imaging (EPI) sequence and includes: 1) use of an inductively coupled implantable coil for improved signal-to-noise ratio (SNR); 2) three-dimensional (3D) automatic shimming of the magnetic field over the spinal cord; and 3) post-acquisition data processing using a multireference scan for minimizing image artifacts. Some of the practical issues in implementing this protocol are discussed. This imaging protocol will be useful in characterizing the spinal cord pathology using techniques that are otherwise time-consuming, such as diffusion tensor imaging.     

In vivo monitoring of gabapentin in rats: a microdialysis study coupled to capillary electrophoresis and laser-induced fluorescence detection

Gabapentin (GP) is a new anticonvulsant used in refractory epilepsy. Few studies have monitored the drug in vivo. We report the combination of capillary electrophoresis and laser-induced fluorescence detection (CZE-LIFD) with brain microdialysis and plasma ultrafiltration in an attempt to measure GP and offer an alternative technique for pharmacokinetic studies. We found that CZE-LIFD is capable of linearly measuring 10(-7)-10(-9) M GP in a 1 nL volume. It was also demonstrated that it is possible to monitor GP in prefrontal cortex dialysates and plasma in rats. It is concluded that the method permits in vivo monitoring of the drug in pharmacological as well as in clinical studies.     

In vivo adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing pancreatic cancer

The vascular endothelial injury with its consequent activation is actively involved in inflammation and promotion of a procoagulant state, which are likely to be of major importance in the pathogenesis of various disorders, including renal thrombotic microangiopathy. This study briefly reviews the consequences of glomerular endothelial cell injury or activation, as shown by recent experimental data.     

The role of imaging in pancreatic cancer

The clinical diagnosis of pancreatic cancer is notoriously difficult, and modern radiological imaging methods are critical to the diagnosis and accurate staging of the disease. Furthermore, endoscopic and interventional radiological techniques have now replaced surgery in palliating those patients with nonresectable disease.     

Determination of kynurenic acid by capillary electrophoresis with laser-induced fluorescence detection

We have investigated the influence of three structurally different but functionally related compounds [1, 10 ortho-phenanthroline (phenanthroline), Rifampicin and aurin tricarboxylic acid (ATA)] on the rate and the extent of proliferation of progesterone-responsive T47D human breast cancer cells. These compounds have previously been used in this laboratory and have been shown to modulate properties of nucleic acid binding proteins. Because p53 and the progesterone receptor (PR) are both DNA binding proteins that appear to regulate proliferation of breast cells, alterations in T47D cell p53 and PR levels were examined to determine their relevance in cell proliferation. T47D cells were grown in the absence of phenol red and in the presence of 5% fetal calf serum with or without charcoal stripping in the presence of the inhibitors. The rate of proliferation of cells grown in Rifampicin containing medium exhibited nearly 70% inhibition. Phenanthroline, a known metal chelator, was an effective inhibitor of proliferation at 3 mM reducing the cell number by more than 75%. ATA (0.24-2.4 micrograms/ml) inhibited the growth of the cells by nearly 50%. Analysis of the mechanism of action of these compounds revealed that treatment with these compounds caused specific changes in the molecular composition of T47D cell PR. Whereas ATA caused increased stability of PR isoforms, Rifampicin induced a upshift in the mobility of PR in SDS gels-a phenomenon associated with hyperphosphorylation of steroid receptors (SRs). Phenanthroline treatment (> 2 mM) caused a complete down-regulation of PR and the tumor suppressor protein, p53. The downregulation of p53 paralleled the changes in the molecular composition of PR. We propose that the inhibition of T47D cell proliferation by phenanthroline, Rifampicin and ATA results from a number of cellular changes that include regulation of p53 and PR.     

In vivo fluorescence detection of human bladder carcinoma without sensitizing agents

The diagnosis of transitional cell bladder carcinoma and especially carcinoma in situ of the bladder in spinal cord disordered persons is often made difficult by catheters, infections or stone-induced chronic inflammation. Fluorescence tagging of tumors by sensitizing agents such as hematoporphyrin derivatives enhances visualization but presents a number of drawbacks for the patients, even at low doses. We have developed a cystoscopic fiber optic instrument, based on a mercury arc lamp, for in vivo detection of human bladder carcinoma without sensitizing agents. The tissue autofluorescence upon UV excitation (365 nm) is detected and a demarcation contrast ratio for carcinoma in situ and transitional cell carcinoma of 2.6 and 3.2 respectively is obtained. This demarcation ratio is 60 percent higher than the contrast ratio obtained after photosensitizer injection. The integration of a reliable diagnostic method with a known efficient therapeutic technique (Nd YAG laser irridiation) opens the way for cost-effective preventive care of high-risk patients.     

In vivo fluorescence kinetics of porphyrins following intravesical instillation of 5-aminolaevulinic acid in normal and tumour-bearing rat bladders

The fluorescence kinetics of protoporphyrin IX (PPIX) following intravesical instillation of 5-aminolaevulinic acid (5-ALA) have been studied in vivo in a rat bladder tumour model. 5-ALA dissolved in NaHCO3 was intravesically instilled for 60 min in tumour-bearing and normal bladders of Wistar rats. The fluorescence was excited with the violet lines of a Kr(+)-laser and recorded in vivo by means of a fibre coupled optical multichannel analyser. The fluorescence emission bands of PPIX at lambda = 636 nm and lambda = 708 nm were detected in normal and tumorous urothelium after only 30 min. The maximum fluorescence intensity was obtained in tumorous and normal urothelium 3-4 h after instillation. The ratio of the fluorescence intensity in tumorous to normal urothelium decreased continuously from four to about two during the time range of 6 h. PPIX fluorescence following 5-ALA instillation could also be observed in kidney and liver. Fluorescence from further porphyrin species with emission bands at lambda = 617 nm and lambda = 682 nm was detected in the bladder, indicating an efflux of hydrophilic porphyrins from the hepatic pathway.     

In vivo study of halothane hepatotoxicity in the rat using magnetic resonance imaging and 31P spectroscopy

Using magnetic resonance imaging (MRI) and spectroscopy (MRS), in vivo halothane hepatotoxicity was assessed in male Wistar rats. With 1.5% halothane in 100 or 20% O2, an edematous region, characterized by increased intensity on T2 weighted images and an increase in regional tissue water content (rho water), was seen proximal to the hepatic portal vein in the liver. Both spin-lattice relaxation (T1) and spin-spin relaxation (T2) increased in this region, relative to distal regions of the liver. Similarly, a high signal intensity on proton density weighted images was observed in this area. As halothane anaesthesia progressed, a decrease in the adenosine triphosphate-inorganic phosphate ratio (ATP/Pi) and an increase in the phosphomonoester-phosphodiester (PME/PDE) ratio was detected in the liver. In addition, intracellular pH decreased and intracellular free magnesium concentration [Mg2+] increased with time of exposure. Excessive vacuolation, ribosomal disappearance from rough endoplasmic reticulum, mitochondrial swelling and fragmentation of smooth endoplasmic reticulum were observed by transmission electron microscopy (TEM) in samples from the edematous region of the liver.     

Assay of trypsin activity by capillary isoelectric focusing with laser-induced fluorescence detection

Capillary isoelectric focusing is a highly effective method for the separation of proteins due to focusing as a function of their pI values in the separation process. This technique is also effective for certain types of peptides that focus well. Fluorescence labeling and subsequent detection by laser-induced fluorescence farther enhance the sensitivity of this technique. This paper demonstrates the utility of this technique in an enzyme assay. A synthetic nona peptide, H-Gly-Cys-His-Glu-Ala-Arg-Ala-Glu-Glu-OH, was labeled with an iodoacetyl derivative of Lissamine rhodamine B at the thiol group of the cysteine residue as a substrate for trypsin. Trypsin catalyzed the cleavage of the Arg-Ala bond of the labeled substrate, which focused at pH 4.8, and liberated a shortened, labeled product, H-Gly-*Cys-His-Glu-Ala-Arg-OH that focused at pH 6.9 (* indicates the label). The product peptide at 3-300 pM was determined with a relative standard deviation of 5.5% (n = 5) by fluorescence detection at 590 nm with excitation by a green line of He-Ne laser. Incubation of trypsin with the substrate for 10 min at 37 degrees C allowed the determination of 50-250 pg of trypsin, with a relative standard deviation of 5.3% (n = 5).     

In vivo heat shock protects rat myocardial mitochondria

Heat shock (HS)/stress proteins (HSP) provide protection from a variety of stresses other than HS, including oxidative stress and mitochondria have been implicated as the target of HS-related protection in stressed cultured cells. Here we investigated whether mitochondria also are targets for the HS-mediated protection in vivo. Sprague Dawley rats were exposed, or not, to HS (41 degrees C, 15 min). After a 21 h recovery period, hearts were excised and perfused with or without H2O2 (0.15 mM). Myocardial mitochondria were then isolated, and their oxygen consumption was analyzed. HS prevented H2O2-induced alterations in state 3 respiration while increasing the expression of Hsp70 and heme oxygenase (HO). Thus, in vivo HS protects rat myocardial mitochondrial respiration against the deleterious effects of oxidative injury, a protection relating to Hsp70 and/or HO and targeting state 3 respiration.     

In vivo glycerol metabolism in the pregnant rat

Pregnant rats at 12 and 21 days of gestation and their virgin controls were injected intravenously with U-14C-glycerol and decapitated 1, 3, or 10 min later. The conversion of labelled glycerol to 14C-glucose was augmented in the 21-day pregnant rats. The disappearance of the newly formed 14C-glucuse from blood was faster in both 12- and 21-day pregnant rats than in their controls, being partially retained as liver 14C-glycogen. The greatest amount of radioactivity in all tissues appeared in the carcass hydrosoluble fraction. This amount was smaller in the pregnant rats. The reduced utilization of glycerol by extrahepatic tissues allowed the 21-day pregnant rats to dispose a greater amount of this substrate for gluconeogenesis.     

In vivo stimulation-induced release of manganese in rat amygdala

The role of manganese ions in neural functions is poorly understood because of the low level of manganese in the brain. An excess of this ion is associated with neurological disorders such as extrapyramidal symptoms. We demonstrated that manganese may be taken up by piriform neurons (tertiary olfactory neurons) after release from the terminals of secondary olfactory neurons, in which 54Mn taken up by the soma may be anterogradely transported [A. Takeda, Y. Kodama, S. Ishiwatari, S. Okada, Manganese transport in the neural circuit of Rat CNS, Brain Res. Bull. (45) (1998) 149-152]. Here we demonstrate for the first time that 54Mn previously taken up into the amygdala is released with neurotransmitters into the extracellular space during stimulation with high K+. The results suggest that the role of manganese ions in the amygdala, and probably in the olfactory system, is dynamically linked to neural signalling processes.     

In vivo acylation of rat brain myelin proteolipid protein

Examination of brain myelin proteins by sodium dodecyl sulfate-gel electrophoresis followed by fluorography clearly showed that both proteolipid protein (PLP) and DM-20 were acylated 24 h after the intracerebral injection of 30-day-old rats with [3H]palmitic acid. The radioactivity associated with PLP remained after purification, re-electrophoresis, and fluorography. Most of the radioactivity associated with PLP was removed when the gels were treated with hydroxylamine and then fluorographed, indicating that fatty acids were bound to PLP by ester linkage. Cleavage of purified PLP with methanolic sodium hydroxide readily released almost all protein-bound radioactivity. Thin layer chromatography of this material on both silver nitrate and reverse-phase plates provided evidence that most of the radioactivity co-migrated with methyl palmitate (77%) and methyl stearate (19%); however, some radioactivity was associated with methyl oleate (4%). Gas-liquid chromatography of the fatty acids associated with PLP distinctly revealed the presence of methyl palmitate and a detectable peak of methyl stearate.     

Single strand conformational polymorphism using capillary electrophoresis with two-dye laser-induced fluorescence detection

Capillary electrophoresis (CE), using a replaceable polymer matrix and two-dye laser-induced fluorescence has been applied to single strand conformational polymorphism (SSCP). Two-dye laser-induced fluorescence has been used for improved strand identification over a single-dye approach. Conditions suitable in the capillary format for rapid separation and high resolution have been explored. The influence of separation parameters such as temperature and matrix composition on separation in SSCP was first determined. Short analysis times allowed for fast screening of optimal separation conditions of the sample. Based on these results, the two strands of a standard 255 bp fragment of the lacI gene were resolved within 25 min with replaceable linear polyacrylamide as a separation matrix. The method was then applied to the detection of different mutations, in the presence of wild type, of a 276 bp fragment of the insulin-like growth factor 1-binding protein 1 (IGF1-BP3) gene.     

Capillary electrophoresis/laser-induced fluorescence detection of fluorescein as a groundwater migration tracer

Capillary electrophoresis (CE) has been applied to the determination of the groundwater migration tracer dye fluorescein based on laser-induced fluorescence (LIF) detection and compared to determinations obtained with traditional spectrofluorimetry. Detection limits of injected dye in the low parts per trillion (ppt) ranges have been accomplished with both CE/LIF based on the Ar ion laser and with a spectrofluorimeter. This approach was used for a real-world problem in determining groundwater migration between adjacent Resource Conservation and Recovery Act (RCRA) and Superfund sites by the Environmental Sciences Division in response to regional needs and as application of new analytical tools under development. Fluorescent dye was injected into source wells and then was determined in monitoring wells by extracting pads that adsorbed the dye or by directly determining the dye in the water using solid-phase extraction (SPE), a preconcentration technique. The approaches based on CE/LIF exhibits increased specificity over existing approaches due to the separation and unique migration time of the dye. Additional studies were aimed at achieving sub-ppt levels in the water using solid-phase extraction and field-amplified injection techniques.     

In vivo behaviour of rat band 3 cross-linked carrier erythrocytes

Inhibitory pathways in the spinal cord play an important role in establishing the pattern of motor discharge. In the wallaby spinal cord preparation, disruption of glycinergic and gamma-amino butyric acid (GABA)ergic neurotransmission abolished the alternation between antagonistic motor pools during fictive locomotion. A new pattern of motor discharge also appeared when both glycine and GABA(A) receptors were blocked simultaneously. This discharge pattern was biphasic, characterized by a distinct pause between two bursts of motoneurone firing during each cycle of motor activity. Whole cell patch recordings showed that the second burst of motor discharge was not caused by a separate inward current at a delayed time course. Furthermore, local injection of an N-methyl-D-aspartic acid (NMDA) specific antagonist converted the biphasic discharge to a continuous burst pattern. The result suggests an NMDA-mediated mechanism, which causes a suppression of motoneurone firing when glutamate release from interneurones is enhanced in the absence of glycinergic and GABAergic inhibition.