Extracting drug mechanism and pharmacodynamic information from clinical electroencephalographic data using generalised semi-linear canonical correlation analysis.

Conventional analysis of clinical resting electroencephalography (EEG) recordings typically involves assessment of spectral power in pre-defined frequency bands at specific electrodes. EEG is a potentially useful technique in drug development for measuring the pharmacodynamic (PD) effects of a centrally acting compound and hence to assess the likelihood of success of a novel drug based on pharmacokinetic-pharmacodynamic (PK-PD) principles. However, the need to define the electrodes and spectral bands to be analysed a priori is limiting where the nature of the drug-induced EEG effects is initially not known. We describe the extension to human EEG data of a generalised semi-linear canonical correlation analysis (GSLCCA), developed for small animal data. GSLCCA uses data from the whole spectrum, the entire recording duration and multiple electrodes. It provides interpretable information on the mechanism of drug action and a PD measure suitable for use in PK-PD modelling. Data from a study with low (analgesic) doses of the µ-opioid agonist, remifentanil, in 12 healthy subjects were analysed using conventional spectral edge analysis and GSLCCA. At this low dose, the conventional analysis was unsuccessful but plausible results consistent with previous observations were obtained using GSLCCA, confirming that GSLCCA can be successfully applied to clinical EEG data.

Vasopressin-induced hypertrophy in H9c2 heart-derived myocytes.

Protein synthesis in H9c2 heart-derived myocytes responds biphasically to arginine vasopressin (1 microM). An initial 50% inhibition attributable to Ca(2+) mobilization from the sarcoplasmic/endoplasmic reticulum is followed by a recovery that subsequently converts to a 1.5-fold stimulation. This study was undertaken to ascertain whether vasopressin programs H9c2 cells to undergo hypertrophy or to proliferate and whether early translational inhibition is required for programming. Translational suppression was observed only at vasopressin concentrations (>1 nM) causing extensive (>50%) depletion of Ca(2+) stores and was diminished at supraphysiologic extracellular Ca(2+) concentrations. Stimulation of protein synthesis, by contrast, was unaffected by changes in extracellular Ca(2+), depended on gene transcription, was suppressed by a protein kinase C pseudosubstrate sequence (peptide 19-27), and was observed at pM vasopressin concentrations. Activation of MAP kinases, phosphoinositide 3-kinase, calcineurin, S6 kinase, or eIF4 could not be implicated in the stimulation, which persisted for 24 h. Vasopressin-treated H9c2 cells underwent hypertrophy by standard criteria. Cellular protein accumulation occurred at pM hormone concentrations, was blocked by peptide 19-27, was observed regardless of retinoic acid pretreatment to prevent myogenic transdifferentiation, and preceded full repletion of Ca(2+) stores. It is proposed that H9c2 cells, which possess all basic features of V1-vasopressin receptor signaling, provide a convenient model for investigating vasopressin-induced myocyte hypertrophy. Early translational suppression is not needed for vasopressin-induced H9c2 myocyte hypertrophy whereas activation of protein kinase C appears essential.

Role of proteolysis and apoptosis in regression of pulmonary vascular remodeling.

Remodeled pulmonary arteries return to normal structural conditions after the increase in pulmonary artery flow resistance is reversed. We studied whether proteolysis of extracellular matrix proteins and apoptosis occur during reversal of remodeling produced by chronic hypoxia in the rat. Main pulmonary arteries were removed at different times during a 10-day period of exposure to 10% O2 and 14 days after return to air. Content and rates of degradation of collagen and elastin as well as immunoreactive collagenase in tissue and isolated mast cells were measured. Immunoblots for collagenase and tissue inhibitor of metalloproteinases (TIMP) were performed. Apoptosis was assessed by cleavage of DNA and TUNEL assay. Excess collagen and elastin present at 10 days of hypoxia decreased to near normal levels after 3-5 days of air. Transient increases in collagenolytic and elastolytic enzyme activities accompanied the rapid decrease in matrix proteins. Mast cells containing collagenase accumulated in remodeled pulmonary arteries, and the active form of collagenase appeared at the time of peak proteolytic activity. TIMP increased during remodeling. Apoptosis was maximal 3 days after return to air. Our results suggest that activation of enzymes, which degrade matrix proteins, and apoptosis play a role in resolution of vascular remodeling.

Expression of matrix-degrading enzymes in pulmonary vascular remodeling in the rat.

Exposure of rats to hypoxia causes pulmonary arterial remodeling, which is partly reversible after return to air. We hypothesized that degradation of excess collagen in remodeled pulmonary arteries in the posthypoxic period is mediated by endogenous matrix metalloproteinases (MMPs). Total proteolytic, collagenolytic, and gelatinolytic activities, levels of stromelysin-1 and tissue inhibitor of metalloprotease-1 (TIMP-1), and immunolocalization of stromelysin-1 in main pulmonary arteries were determined after exposure of rats to 10% O2 for 10 days followed by normoxia. We observed transient increases in total proteolytic, collagenolytic, and gelatinolytic activities and expression of approximately 72-, 68-, and 60-kDa gelatinases by zymography within 3 days of cessation of hypoxic exposure. The level of TIMP-1 increased as the stromelysin-1 level increased. Immunoreactive stromelysin-1 was localized predominantly in the luminal region of normal and hypertensive pulmonary arteries. These results are consistent with the notion that endogenous MMPs may mediate the breakdown of excess collagen in remodeled pulmonary arteries during the early posthypoxic period.

Mast cell collagenase correlates with regression of pulmonary vascular remodeling in the rat.

Pulmonary vascular remodeling, produced by cell hypertrophy and extracellular matrix protein synthesis in response to hemodynamic stress, regresses after reduction of blood pressure, possibly by proteolysis of structural proteins. To test this postulate, we assessed the breakdown of extracellular matrix proteins and expression of collagenase and elastase in pulmonary arteries of rats exposed to hypoxia (10% O2 for 10 d) followed by normoxia. During hypoxia, contents of collagen and elastin increased in pulmonary arteries and latent rat interstitial collagenase was expressed without increased collagenolytic activity or mRNA levels. At 3 days after normoxia, collagen and elastin contents decreased coincident with the new appearance of activated collagenase and transient increases in collagenolytic and elastolytic activities. The amount of immunoreactive collagenase, localized predominately in connective tissue-type mast cells, was increased in the adventitia and media of hypertensive vessels. We conclude that mast cells containing latent collagenase are recruited into the outer walls of pulmonary arteries during remodeling. It is possible that mast cell-derived collagenase contributes to collagen breakdown in pulmonary arteries during early recovery from hypoxia and plays a role in restoration of vascular architecture.

Liposome-entrapped antifibrotic agent prevents collagen accumulation in hypertensive pulmonary arteries of rats.

We studied the therapeutic efficacy of an intravenously injected antifibrotic agent encapsulated in liposomes on inhibiting collagen accumulation in hypertensive blood vessels. cis-4-Hydroxy-L-proline (cHyp) in liposomes was injected into rats exposed to 10% O2, and drug effect was evaluated by measuring right ventricular pressure and hydroxyproline content of the pulmonary artery. Right ventricular pressure was 11 +/- 1 mm Hg (mean +/- SEM) 5 days after a single intravenous injection of 200 mg/kg cHyp in liposomes compared with 14 +/- 1 mm Hg in rats injected with empty liposomes; hydroxyproline content was also reduced by cHyp treatment (87 +/- 6 versus 107 +/- 7 micrograms per vessel) (p less than 0.05 for both, n = 6-9). Injections of cHyp in liposomes every 5 days partially prevented hypertension and vascular collagen accumulation during a 3-week exposure to hypoxia, and the dose required was one tenth the dose of unencapsulated cHyp. Therapeutic doses of cHyp in liposomes injected for 6 months affected tensile properties of main pulmonary artery and aorta, but there were no apparent histological effects on other organs. Liposomes injected intravenously were identified in pulmonary artery endothelial cells. The prolonged effect of a single injection of cHyp in liposomes may be due to uptake of the liposomes by the endothelium. Liposome delivery of drugs to the arterial wall may be useful in the study and treatment of hypertensive vascular disease.

The effects of nerve growth factor and dibutyryl cyclic AMP on cytoskeletal densities in cultured sensory ganglia.

The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths. As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in vitro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofilaments was seen at 24 hr, with equal densities of both cytoskeletal elements present after 48 hr in vitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC, implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neurites of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.

Twice-daily tapering dexamethasone treatment during cranial radiation for newly diagnosed brain metastases.

Twenty evaluable patients with newly diagnosed brain metastases underwent treatment with a novel dose/schedule of dexamethasone aimed at reducing steroid toxicity during palliative radiation therapy. All patients received twice daily dexamethasone starting at 8 mg bid for four days then 4 mg bid for four days then 2 mg bid until the last day of radiation therapy. The radiation prescriptions were not standardized varying from 2000 cGy/5 fractions to 5800 cGy/29 fractions. Fourteen patients received dexamethasone for a minimum of 24 hours before their first radiation treatment and 7 (50%) experienced improvement in neurologic symptoms/signs prior to starting radiation treatments. Fourteen patients completed the planned course of radiation and dexamethasone. Only 1 patient needed to restart dexamethasone within 30 days of finishing radiation because of steroid reversible neurologic deficits. Steroid toxicity was mild including hyperglycemia (1), candida esophagitis (1), steroid pseudorheumatism (2), peripheral edema (1) and steroid withdrawal syndrome (1). Only two toxic events were recorded in patients receiving steroids less than 21 days. Twice daily dexamethasone appears to provide good clinical results with minimal morbidity.

Neuropeptide Y-containing nerves in rat gonads: sex difference and development.

The objectives of the present study were 1) to evaluate for a sex difference in innervation of adult rat gonads by neuropeptide Y-immunoreactive (NPY-I) nerves and 2) to examine the development of innervation of rat gonads by NPY-I nerves during the fetal and neonatal periods. With fluorescence immunocytochemistry, NPY-I nerves were profuse in adult ovarian tissues. Ovarian blood vessels were particularly well innervated by NPY-I nerves, and nerves were also detected in interstitial gland tissues. No nerves were found within the testis, and NPY-I nerves were only rarely located within the tunica albuginea. During fetal life, ovaries were devoid of NPY-I nerves; however, nerves were visualized within the connective tissue immediately peripheral to the ovary on fetal Day 22. As early as postnatal Day 2, NPY-I nerves were observed in connective tissue septa of the developing ovary. By postnatal Day 12, NPY-I nerves surrounded developing follicles and blood vessels of the ovarian cortex. In the developing testis after postnatal Day 5, NPY-I nerves were limited to the tunica albuginea and surrounding large subcapsular blood vessels. Structures within the testis lacked innervation by NPY-I nerves. These anatomical studies suggest that NPY-I nerves are absent in the gonads during fetal life and grow into the ovary and not the testis during the perinatal period and that NPY-I nerves may play a role in the functioning of the rat ovary, but may not be important in control of testicular function.

Calmodulin-dependent phosphatases of PC12, GH3, and C6 cells: physical, kinetic, and immunochemical properties.

Calmodulin-dependent phosphoprotein phosphatase (CaMDP) activity has been found in each of three cultured cell lines: rat pheochromocytoma (PC12), glioma (C6), and pituitary adenoma (GH3) cells. These CaMDP activities bind to immobilized calmodulin in the presence of Ca2+ and are eluted by EGTA. Sucrose density centrifugation revealed that the phosphatase activities exhibited sedimentation coefficients of 4.37, 4.23, and 4.59 for proteins derived from C6, GH3, and PC12 cells, respectively. The Stokes radii measured for the PC12 and C6 activities were 41.8 and 40.0 A, respectively. The estimated molecular weights calculated for the enzymes from these data are 79,100 and 72,200. The phosphatase activities required the presence of divalent cations such as Ca2+ or Mn2+ for expression of activity, which was optimal only in the presence of calmodulin. The apparent Km for phosphorylated myelin basic protein substrate was 8 microM. Affinity-purified antibodies to the B subunit of bovine brain CaMDP were found by immunoblot (Western blot) to cross-react with a single protein among proteins extracted from PC12, C6, and GH3 cells that had been resolved by two-dimensional electrophoresis. In each case, the cross-reacting protein exhibited an Mr of 16,000 and an isoelectric point of 4.7, values virtually identical to those reported previously for the B subunit of bovine brain CaMDP (sometimes called calcineurin). This cross-reacting protein was found among cellular proteins eluted from immobilized calmodulin by EGTA. Immunocytochemical localization of the cross-reacting protein in undifferentiated PC12 cells or in cells differentiated in response to nerve growth factor revealed its presence diffusely throughout the cytoplasm. These experiments support the contention that each of these cell lines contains a calmodulin-regulated phosphatase homologous physically and kinetically, and immunologically related to bovine brain CaMDP.

Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

Sleep apnea and the Arnold-Chiari malformation.

We present a polysomnogram-documented report of central sleep apnea (427 events) and moderately severe decreases in arterial oxygen saturation (to 81%) associated with the Arnold-Chiari malformation (ACM). Daytime hypersomnolence and other symptoms had significantly impaired our patient's work performance. After surgical correction of the ACM, there was marked improvement in symptomatology. A post-surgery polysomnogram revealed marked improvement in the number of central apneas (74 events) and only mild decreases in oxygen saturation (to 94%).

Quantitative deposition of ultrafine stable particles in the human respiratory tract.

Theoretical models of particle deposition in the respiratory tract predict high fractional deposition for particles of less than 0.1 micron, but there are few confirming experimental data for those predictions. We have measured the deposition fraction of a nonhygroscopic aerosol in the human respiratory tract. The aerosol had a count mean diameter of 0.044 micron SD of 1.93, as measured with an electrical aerosol analyzer, and was produced from a 0.01% solution of bis(2-ethylhexyl) sebacate using a condensation generator. Subjects inhaled the aerosol using a controlled respiratory pattern of 1 liter tidal volume, 12/min. Deposition was calculated as the difference in concentration between inhaled and exhaled aerosol of five size fractions corrected for system deposition and dead-space constants. Three deposition studies were done on each of five normal male volunteers. Means (+/- SE) for the five size fractions were 0.024 micron, 0.71 +/- 0.06; 0.043 micron, 0.62 +/- 0.06; 0.075 micron, 0.53 +/- 0.05; 0.13 micron, 0.44 +/- 0.04; and 0.24 micron, 0.37 +/- 0.06. These data demonstrate that deposition of inhaled particles in the 0.024- to 0.24-micron size range is high and increases with decreasing size. These observations agree with and validate predictions of mathematical models.

Concentration and particle size distribution in smoke from marijuana cigarettes with different delta 9-tetrahydrocannabinol content.

Particle size and mass concentration are important determinants of site and quantity of respiratory tract deposition of aerosols. Particle concentrations and size distributions of smoke from marijuana cigarettes with different concentrations (as measured in the marijuana leaf) of delta 9-tetrahydrocannabinol (delta 9-THC) were measured using a single particle aerodynamic relaxation time (SPART) analyzer. The SPART analyzer measures aerodynamic diameter of single suspended particles at a rate of 3000/min. Cigarettes were smoked using a 35-cc, 2-sec puffing device attached to a diluter; dilution and analysis were completed within 4 sec of puff generation. The size distribution of smoke from all marijuana cigarettes was similar to that for tobacco cigarettes, ranging from 0.35 to 0.43 micron (count median aerodynamic diameter). The particle number and mass concentration increased as delta 9-THC concentration increased, being, respectively, 2.2- and 3.8-fold higher in the marijuana cigarette leaf with highest delta 9-THC concentration compared to the placebo marijuana cigarette. These data indicate the need for quantitative comparisons of other potentially toxic constituents in marijuana cigarettes of different delta 9-THC concentrations.

A method for exposing the internal anatomy of small and delicate tissues for correlated SEM/TEM studies using polyethylene glycol embedding.

A method for preparing and handling large, clean, distortion-free cut surfaces through small and delicate tissues for correlated SEM/TEM examination is described. In this method, tissues are fixed according to conventional protocols; however, instead of critical-point-drying after fixation, tissues are first embedded in polyethylene glycol (PEG), a water-soluble waxy solid. Tissue blocks are easily oriented and sectioned to the desired regions, immersed in a solvent to remove PEG, critical-point-dried, and examined with an SEM. The same tissue blocks can be reworked for TEM by immersing in propylene oxide and embedding in an epoxy resin.