Biomedical subjects

R R Stewart

Publications and source records attributed to R R Stewart.

At least 19 recordsLinked to original sources

PubMed2002

Neural progenitor cells of the neonatal rat anterior subventricular zone express functional GABA(A) receptors.

The interneurons of the olfactory bulb arise from precursor cells in the anterior part of the neonatal subventricular zone, the SVZa, and are distinctive in that they possess a neuronal phenotype and yet undergo cell division. To characterize the differentiation of neonatal SVZa progenitor cells, we analyzed the complement of ionotropic neurotransmitter receptors that they express in vitro. For this analysis, we tested the sensitivity of SVZa progenitor cells to gamma-amino-n-butyric acid (GABA), adenosine triphosphate (ATP), kainate, N-methyl-D-aspartate (NMDA), and acetylcholine (ACh) after 1 day in vitro. SVZa progenitor cells had chloride currents activated by GABA and muscimol, the GABA(A) receptor-specific agonist, but were insensitive to ATP, kainate, NMDA, and ACh. In addition, GABA- or muscimol-activated chloride currents were blocked nearly completely by 30 microM bicuculline, the GABA(A) receptor-specific antagonist, suggesting that GABA(B) and GABA(C) receptors are absent. Measurements of the chloride reversal potential by gramicidin-perforated patch clamp revealed that currents generated by activation of GABA(A) receptors were inward, and thus, depolarizing. A set of complementary experiments was undertaken to determine by reverse transcription and polymerase chain reaction (RT-PCR) whether SVZa progenitor cells express the messenger RNA (mRNA) coding for glutamic acid decarboxylase 67 (GAD67), used in the synthesis of GABA and for GABA(A) receptor subunits. Both postnatal day (P0) SVZa and olfactory bulb possessed detectable mRNA coding for GAD67. In P0 SVZa, the GABA(A) receptor subunits detected with RT-PCR included alpha 2-4, beta 1-3, and gamma 2S (short form). By comparison, the P0 olfactory bulb expressed all of the subunits detectable in the SVZa and additional subunit mRNAs: alpha 1, alpha 5, gamma 1, gamma 2L (long form), gamma 3, and delta subunit mRNAs. Antibodies recognizing GABA, GAD, and various GABA(A) receptor subunits were used to label SVZa cells harvested from P0-1 rats and cultured for 1 day. The cells were immunoreactive for GABA, GAD, and the GABA(A) receptor subunits alpha 2-5, beta 1-3, and gamma 2. To relate the characteristics of GABA(A) receptors in cultured SVZa precursor cells to particular combinations of subunits, the open reading frames of the dominant subunits detected by RT-PCR (alpha 2-4, beta 3, and gamma 2S) were cloned into a mammalian cell expression vector and different combinations were transfected into Chinese hamster ovary-K1 (CHO-K1) cells. A comparison of the sensitivity to inhibition by zinc of GABA(A) receptors in SVZa precursor cells and in CHO-K1 cells expressing various combinations of recombinant GABA(A) receptor subunits suggested that the gamma 2S subunit was present and functional in the GABA(A) receptor chloride channel complex. Thus, SVZa precursor cells are GABAergic and a subset of the GABA(A) receptor subunits detected in the olfactory bulb was found in the SVZa, as might be expected because SVZa progenitor cells migrate to the bulb as they differentiate.

Animals↗

PubMed2001

Involvement of non-NMDA receptors in the rescue of weaver cerebellar granule neurons and sensitivity to ethanol of cerebellar AMPA receptors in oocytes.

The cellular mechanism responsible for the death of cerebellar granule neurons in the weaver mutant mouse is still being intensely investigated. To determine if alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors are involved in producing the weaver phenotype or are altered by the weaver gene, we used (1) reverse transcription and polymerase chain reaction (RT-PCR) to detect transcripts of glutamate receptors (GluR1-4) from wild-type and mutant cerebella; (2) immunocytochemistry to establish the types of glutamate receptors present in granule neurons cultured from normal and homozygous weaver postnatal day 5-6 (P5-6) cerebella; (3) 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a blocker of glutamate (AMPA/Kainate/NMDA) receptors, and 6,7-dinitroquinoxaline-2,3-dione (NBQX), a blocker of AMPA and kainate receptors, to assess the number of neurons and the number of neurons with long neurites in cultures of homozygous weaver granule neurons; (4) two-electrode voltage clamp recordings to study AMPA glutamate receptor expression in Xenopus oocytes after injection of mRNA isolated from cerebella of normal and weaver P5-6, postnatal day 10 (P10) and postnatal day 23 (P23) mice; and (5) ethanol, which at low 1-10 mM concentrations had been shown previously to rescue homozygous weaver granule neurons in culture [Liesi et al., J. Neurosci. Res. 48 (1997) 571-579], to examine its effect on modulation of AMPA receptors expressed from mRNA. By RT-PCR, the mRNA coding for AMPA receptor subunits GluR1-4 were detected from +/+ and wv/wv cerebella, and by immunocytochemistry, GluR1, GluR2/3 and GluR4 were observed to be expressed in cultured +/+ and wv/wv granule cells. CNQX at 10 microM or NBQX at 10 microM significantly increased the number of surviving neurons and the number with long neurites as compared to wv/wv controls. In addition, CNQX was significantly more effective than NBQX. In oocytes injected with mRNA from P10 normal or weaver cerebella, the amplitudes of the responses to kainate were about equal. In contrast, the amplitudes of the kainate-activated currents in oocytes injected with weaver P23 mRNA were about twice as large as the currents observed in oocytes injected with mRNA from normal P23 cerebella, and both were larger than kainate-activated currents observed after injection of P10 normal and weaver mRNA. Kainate-activated AMPA receptor currents in oocytes injected with mRNA from P10 and P23 normal and homozygous weaver cerebella were inhibited by ethanol. There were no significant differences in the inhibition produced by ethanol on currents from P10 or P23 normal and wv/wv mRNA. Thus, P23 weaver cerebellar mRNA expressed more kainate-activated current in oocytes than P23 normal cerebellar mRNA; both normal and weaver cerebellar granule neurons express mRNA coding for functional AMPA receptors that are susceptible to ethanol inhibition.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2001

Neurons and glial cells of the embryonic human brain and spinal cord express multiple and distinct isoforms of laminin.

We have identified by immunocytochemistry, Western blotting, and RT-PCR the isoforms of laminin expressed by glial cells and neurons cultured from human embryonic brain and spinal cord. We show that most of the known laminins are present in human neurons and glial cells. Importantly, Western analysis demonstrates that the isoforms of laminin present in embryonic human brain differ from those expressed in human spinal cord. Neurons of the brain and spinal cord also express their distinct and characteristic isoforms of laminin compared to the glial cells of the same CNS regions. These results suggest that, in addition to the known laminins, several novel isoforms may exist in the human embryonic CNS. The observed differences between the isoforms of laminin in brain and spinal cord neurons and glial cells may result from primary structural changes or from posttranslational modifications, e.g., variations in glycosylation. Thus, identification of these novel laminins and determination of their function(s) should further our understanding of the mechanisms of aging, disease, and trauma in the human CNS.

Animals↗

PubMed2001

Biochemical, phenotypic and neurophysiological characterization of a genetic mouse model of RSH/Smith--Lemli--Opitz syndrome.

The RSH/Smith--Lemli--Opitz syndrome (RSH/SLOS) is a human autosomal recessive syndrome characterized by multiple malformations, a distinct behavioral phenotype with autistic features and mental retardation. RSH/SLOS is due to an inborn error of cholesterol biosynthesis caused by mutation of the 3 beta-hydroxysterol Delta(7)-reductase gene. To further our understanding of the developmental and neurological processes that underlie the pathophysiology of this disorder, we have developed a mouse model of RSH/SLOS by disruption of the 3 beta-hydroxysterol Delta(7)-reductase gene. Here we provide the biochemical, phenotypic and neurophysiological characterization of this genetic mouse model. As in human patients, the RSH/SLOS mouse has a marked reduction of serum and tissue cholesterol levels and a marked increase of serum and tissue 7-dehydrocholesterol levels. Phenotypic similarities between this mouse model and the human syndrome include intra-uterine growth retardation, variable craniofacial anomalies including cleft palate, poor feeding with an uncoordinated suck, hypotonia and decreased movement. Neurophysiological studies showed that although the response of frontal cortex neurons to the neurotransmitter gamma-amino-n-butyric acid was normal, the response of these same neurons to glutamate was significantly impaired. This finding provides insight into potential mechanisms underlying the neurological dysfunction seen in this human mental retardation syndrome and suggests that this mouse model will allow the testing of potential therapeutic interventions.

Animals↗

PubMed2001

Characteristics of endothelial cells derived from the blood-brain barrier and of astrocytes in culture.

In this study, cultures of astrocytes and capillary endothelial cells from the blood-brain barrier (BBB) of the postnatal (P1) mouse cerebral cortex were analyzed with the aim of acquiring information on the distinguishing characteristics of each cell type. For isolation and purification of astrocyte cells, the methods of McCarthy and DeVellis [J. Cell Biol. 85 (1980) 890] were employed. The methods of Chen et al. [Lab. Invest. 78 (1998) 353], Duport et al. [Proc. Natl. Acad. Sci. USA 95 (1998) 1840], Rubin et al. [J Cell Biol. 115 (1991) 1725] and Tontsch and Bauer [Microvasc. Res. 37 (1989) 148] were utilized for culturing of cells from the BBB. A simple protocol was also created for isolating and purifying brain endothelial cells with 10 mM sodium cyanide. The vascular system of the cerebral cortex is derived from the leptomeningeal blood vessels [Qin and Sato, Dev. Dyn. 202 (1995) 172; Risau et al., EMBO J. 5 (1986) 3179]. With this in mind, cultures of the P1 mouse meninges were used as a comparative cell type in order to differentiate between BBB cells and astrocytes. In this regard, the expression of a number of markers were correlated, and an antibody double labeling technique was employed. The staining of these markers was then compared to cells cultured from leptomeninges and to two other types of endothelial cells, human umbilical vein and bovine aortic. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA isolated from adult mouse brain, cells cultured from P1 mouse cortex or meninges, bovine aortic endothelial cells and human umbilical vein endothelial cells (HUV-EC) to detect the expression of glial fibrillary acidic protein (GFAP), Von Willebrand factor (factor VIII-related antigen) and fibronectin. These analyses revealed the presence of GFAP mRNA in the cultures of cortical and leptomeningeal cells and of protein in all cell types; Von Willebrand factor mRNA was detectable in HUV-EC cells but undetectable in cortical, leptomeningeal and bovine aortic endothelial cells. Fibronectin mRNA and protein were present in all of the cell types. Given the results of our investigations we conclude that in culture, astrocytes are actually brain endothelial cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Alcohols inhibit N-methyl-D-aspartate receptors via a site exposed to the extracellular environment.

N-Methyl-D-aspartate (NMDA) receptors are important CNS target sites of alcohols, but the site and mechanism of action of alcohols on NMDA receptors remains unclear. In CHO-K1 cells transfected with NR1/NR2B NMDA receptor subunits, ethanol inhibited NMDA-activated current with an IC(50) of 138 mM. Truncation of the intracellular C-terminal domain of the NR1 subunit (NR1T) did not alter ethanol sensitivity when combined with the NR2B subunit, but a similar truncation of the NR2B subunit (NR2BT) slightly enhanced ethanol sensitivity of receptors formed from coexpression with either NR1 or NR1T subunits. 1-Pentanol applied externally inhibited NMDA receptors with an IC(50) of 9.9 mM, but intracellular application of 1-pentanol (25 mM) did not alter NMDA receptor inhibition by externally applied ethanol or 1-pentanol. In addition, the amplitude of NMDA-activated current did not decrease during the time required for 1-pentanol (25 mM) to diffuse throughout the cytoplasm. Ethanol did not inhibit NMDA receptors when bath-applied in cell-attached patches or when applied to the cytoplasmic face of inside-out membrane patches. These results appear to be best explained by an action of alcohols on the NMDA receptor-channel protein, at a site located in a domain exposed to, or only accessible from, the extracellular environment.

Animals↗

PubMed2000

Involvement of GIRK2 in postnatal development of the weaver cerebellum.

We demonstrate that the homozygous weaver granule neurons cultured on a laminin substratum fail to express inwardly rectifying potassium currents, including a functional G-protein coupled inwardly rectifying potassium (GIRK)2 potassium channel. By contrast, both normal and weaver Purkinje cells express inwardly rectifying potassium currents, and normal granule cells exhibit inwardly rectifying potassium currents inducible with GTP-gamma-S. In protein extracts of the vermal postnatal day (P)5-9 weaver cerebellum, the GIRK2 protein could not be detected by Western analysis, although the GIRK2 protein was detectable in extracts of the normal vermis. Northern analysis indicated that during early postnatal cerebellar development, the GIRK2 mRNA is expressed at extremely low levels being detectable at P18-23 in the normal but not yet in the homozygous weaver cerebellum. Using reverse transcriptase-polymerase chain reaction (RT-PCR), the GIRK2 mRNA was detected in both normal and weaver cerebella, but quantitative PCR confirmed that the weaver cerebellum expressed the GIRK2 gene at significantly lower levels as compared to the normal cerebellum (P = 0.01, paired t-test). Sequencing indicated that the weaver GIRK2 channel gene had the point mutation proposed to be responsible for the weaver phenotype. Rescue of both survival and neurite outgrowth of the cultured vermal weaver granule neurons by verapamil (Liesi and Wright, 1996; Liesi et al., 1999) induced expression of immunocytochemically detectable levels of the GIRK2 protein. Sequencing revealed that the GIRK2 mRNA of the rescued weaver granule neurons remained the mutated variant of the GIRK2 channel gene. Our results indicate that expression of the mutated GIRK2 protein and/or mRNA in the weaver granule neurons may be an indicator of rescue rather than death of the weaver granule neurons. That the weaver granule neurons expressed no functional GIRK2 receptors during a time period of neuronal death and migration failure suggests that the point mutation in the H5 membrane spanning region of the GIRK2 gene may associate with, but not be responsible for the weaver phenotype.

Animals↗

PubMed1999

Potassium currents in precursor cells isolated from the anterior subventricular zone of the neonatal rat forebrain.

The progenitor cells from the anterior part of the neonatal subventricular zone, the SVZa, are unusual in that, although they undergo division, they have a neuronal phenotype. To characterize the electrophysiological properties of the SVZa precursor cells, recordings were made of potassium and sodium currents from SVZa cells that were removed from postnatal day 0-1 rats and cultured for 1 day. The properties of the delayed rectifier and A-type potassium currents were described by classical Hodgkin and Huxley analyses of activation and inactivation. In addition, cells were assessed under current clamp for their ability to generate action potentials. The A-type potassium current (IK(A)) was completely inactivated at a holding potential of -50 mV. The remaining potassium current resembled the delayed rectifier current (IK(DR)) in that it was blocked by tetraethylammonium (TEA; IC50 4.1 mM) and activated and inactivated slowly compared with IK(A). The conductance-voltage (G-V) curve revealed that G increased continuously from 0.2 nS at -40 mV to a peak of 2.6 nS at +10 or +20 mV, and then decreased for voltages above +30 mV. Activation time constants were largest at -40 mV ( approximately 11 ms) and smallest at 100 mV ( approximately 1.5 ms). The properties of IK(A) were studied in the presence of 20 mM TEA, to block IK(DR), and from a holding potential of -15 mV, to inactivate both IK(DR) and IK(A). IK(A) was then allowed to recover from inactivation to negative potentials during 200- to 800-ms pulses. Recovery from inactivation was fastest at -130 mV ( approximately 21 ms) and slowest at -90 mV ( approximately 135 ms). Inactivation was voltage independent from -60 to +60 mV with a time constant of approximately 15 ms. At steady state, IK(A) was half inactivated at -90 mV. GK(A) increased from 0.2 nS at -60 mV to a peak of 2.4 nS at +40 mV. Finally, the activation time constants ranged from approximately 1.9 ms at -50 mV to 0.7 ms at +60 mV. The properties of IK(A) resembled those of IK(A) found in differentiating cerebellar granule neurons. Most SVZa cells had sodium currents (28/32 cells). However, in current clamp 11 of 12 cells were incapable of generating action potentials from voltages of -30 to -100 mV, suggesting that the available current densities were too low to support excitability.

Animals↗

PubMed1999

Differential modulation by copper and zinc of P2X2 and P2X4 receptor function.

Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function. The modulation by Cu2+ and Zn2+ of P2X2 and P2X4 receptors expressed in Xenopus oocytes was studied with the two-electrode, voltage-clamp technique. In oocytes expressing P2X2 receptors, both Cu2+ and Zn2+, in the concentration range 1-130 microM, reversibly potentiated current activated by submaximal concentrations of ATP. The Cu2+ and Zn2+ concentrations that produced 50% of maximal potentiation (EC50) of current activated by 50 microM ATP were 16.3 +/- 0.9 (SE) microM and 19.6 +/- 1.5 microM, respectively. Cu2+ and Zn2+ potentiation of ATP-activated current was independent of membrane potential between -80 and +20 mV and did not involve a shift in the reversal potential of the current. Like Zn2+, Cu2+ increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally effective concentration of Zn2+, suggesting a common site or mechanism of action of Cu2+ and Zn2+ on P2X2 receptors. For the P2X4 receptor, Zn2+, from 0.5 to 20 microM enhanced current activated by 5 microM ATP with an EC50 value of 2.4 +/- 0.2 microM. Zn2+ shifted the ATP concentration-response curve to the left in a parallel manner, and potentiation by Zn2+ was voltage independent. By contrast, Cu2+ in a similar concentration range did not affect ATP-activated current in oocytes expressing P2X4 receptors, and Cu2+ did not alter the potentiation of ATP-activated current produced by Zn2+. The results suggest that Cu2+ and Zn2+ differentially modulate the function of P2X2 and P2X4 receptors, perhaps because of differences in a shared site of action on both subunits or the absence of a site for Cu2+ action on the P2X4 receptor.

Adenosine Triphosphate↗

PubMed1999

Weaver cerebellar granule neurons show altered expression of NMDA receptor subunits both in vivo and in vitro.

Biochemical, immunocytochemical, and molecular biological techniques were used to investigate the expression of N-methyl-D-aspartate (NMDA) receptor subunits in migration-deficient weaver mouse cerebellum in vivo and in primary cultures of the vermal weaver granule neurons with or without a rescue by verapamil. We found that both NMDAR1(zeta1) message and protein were expressed by the weaver granule neurons in situ. Immunocytochemical and biochemical analyses indicated that granule neurons of the weaver cerebellum expressed R1(zeta1) and R2A(epsilon1) subunits but showed little expression of the R2B(epsilon2) subunit. In weaver cerebellum, the R2B(epsilon2) subunit was primarily expressed in nerve fibers of the internal granule cell layer and white matter. Reverse-transcriptase-polymerase chain reaction followed by sequence analysis of the R1(zeta1) subunit indicated that the zeta1 subunit amplicons of both normal and weaver cerebella were identical, and that splice variants with exon 22 (1-2) and with or without exon 5 (a/b) or exon 21 (1-4) were detectable. The R2A(epsilon1), and R2B(epsilon2) subunits of the normal and weaver mouse cerebellum revealed no primary structural differences between the normal and weaver NMDA receptor subunits or the cloned mouse NMDA receptor subunits. In vermal cultures, normal granule neurons expressed all three NMDA receptor subunits (zeta1, epsilon1, and epsilon2), whereas the weaver neurons failed to express the epsilon2 subunit. Rescue of the weaver neurons by verapamil induced expression of the epsilon2 protein along the granule neuronal surfaces. The present results suggest that lack of the epsilon2 subunit in the weaver cerebellum may relate to the lack of functional NMDA receptors and/or to the migratory failure of the weaver granule neurons. Our data further suggest that NMDA receptor-mediated neurotoxicity is an unlikely mediator of neuronal death of the weaver granule neurons. In fact, down-regulation of the NMDA receptor expression and function may be a protective measure of the weaver granule neurons to reduce calcium entry via these receptors.

Animals↗

PubMed1998

Phosphorylation of the GABAA receptor gamma2L subunit in rat sensory neurons may not be necessary for ethanol sensitivity.

The effect of ethanol on the current activated by 2.5 to 40 microM gamma-aminobutyric acid (GABA) was studied in freshly isolated rat dorsal root ganglion (DRG) neurons under voltage clamp in the whole-cell and perforated-patch recording configurations. Our results confirmed that GABAA-activated current in these neurons was insensitive to ethanol at concentrations from 2.5 to 100 mM [G. White, D.M. Lovinger, F.F. Weight, Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron, Brain Res. 507 (1990) 332-336.]. In addition, the ethanol sensitivity of GABA receptors was studied under conditions that promote phosphorylation of the PKC site on the gamma2L subunit. The presence of the gamma2L and other subunit mRNAs was detected by reverse transcription (RT) of total RNA purified from adult DRG followed by polymerase chain reaction (PCR) using subunit specific primer sets. We found that the GABA response remained insensitive to 2.5-100 mM ethanol despite: (i) the extracellular preapplication of 5, 20 or 500 nM phorbol 12-myristate 13-acetate (PMA); (ii) raising free intracellular Ca2+ ([Ca2+]i) from 7 to 100 or 600 nM by altering the intracellular Ca2+/EGTA ratio; (iii) intracellular application of PKC (0.247 U ml-1 ); and (iv) combining the intracellular application of 1 microM okadaic acid and 30 microM peptide 3 with the extracellular application of 20 nM PMA. These results suggest that phosphorylation of the gamma2L subunit is not the only requirement for ethanol sensitivity of GABAA receptors.

Animals↗

PubMed1997

Neuronal progenitor cells derived from the anterior subventricular zone of the neonatal rat forebrain continue to proliferate in vitro and express a neuronal phenotype.

A discrete area of the anterior part of the subventricular zone, or SVZa, of the postnatal forebrain is composed of progenitor cells that are dissimilar to those elsewhere in the CNS. In vivo SVZa progenitor cells retain the ability for division, even though they are phenotypically neurons. To characterize further the properties of SVZa cells, we have analyzed their characteristics in vitro using cell-type specific antibodies and their proliferative capacity by the incorporation of bromodeoxyuridine. At 2 h in vitro, as well as after 1 day in vitro, virtually all SVZa cells isolated from the neonatal forebrain express TuJ1, an antibody that recognizes neuron-specific tubulin, and are GFAP-negative. Likewise, the preponderance of SVZa cells express the neuron-specific markers N-CAM and MAP-2 when examined after 1 day in culture. The majority of SVZa cells cultured for as long as 8 days also possessed a neuronal phenotype. In addition, process-bearing TuJ1-positive SVZa cells continued to proliferate throughout the entire culture period. Thus, the neuronal progenitor cells of the SVZa constitute a unique cell population with characteristics distinct from the cells of other germinal zones.

Animals↗

PubMed1997

Volatile general anaesthetic actions on recombinant nACh alpha 7, 5-HT3 and chimeric nACh alpha 7-5-HT3 receptors expressed in Xenopus oocytes.

The effect of halothane and isoflurane was studied on the function of recombinant neurotransmitter receptors expressed in Xenopus oocytes. Both anaesthetics inhibited nicotinic acetylcholine type alpha 7 (nACh alpha 7) receptor-mediated responses, potentiated 5-hydroxytryptamine type 3 (5-HT3) receptor-mediated responses at low agonist concentrations, and inhibited the function of a chimeric receptor (with the N-terminal domain from the nACh alpha 7 receptor and the transmembrane and C-terminal domains from the 5-HT3 receptor) in a manner similar to that of the nACh alpha 7 receptor. Since the N-terminal domain of the chimeric receptor was from the nACh alpha 7 receptor, the observations suggest that the inhibition involves the N-terminal domain of the receptor.

Anesthetics, Inhalation↗

PubMed1996

Mechanisms of isoniazid release from poly(d,l-lactide-co-glycolide) matrices prepared by dry-mixing and low density polymeric foam methods.

The release mechanisms of a small molecular drug from biodegradable poly(d,l-lactide-co-glycolide) (PLGA) cylindrical matrices were investigated. Isoniazid (INH), one of the most effective drugs against tuberculosis (TB), was selected as the model drug. Controlled-release matrices consisting of the drug and polymer were fabricated by two methods. The first of these, the dry-mixing method, involved the extrusion of a mixture of micronized drug and polymer particles as rods. In the second technique, the low density polymeric foam method, drug particles were enclosed in the cells of porous polymeric foams prior to extrusion. In vitro, the dry-mixed matrices released INH more rapidly than the polymeric foam matrices. The Roseman-Higuchi diffusion model, which had previously been found to be effective in analyzing the release kinetics of INH from the dry-mixed matrices, also fit the kinetics of INH released from matrices prepared from polymeric foams. This indicated that the release was still diffusion-controlled rather than degradation-controlled. The release mechanisms were further investigated, and two diffusion mechanisms, pore diffusion and lattice diffusion, were proposed for the INH controlled-release matrices according to the way in which they were prepared. Matrices prepared by the dry-mixing method appear to segregate drug particles along polymer grain boundaries and thus have a pore diffusion mechanism, while matrices prepared by the foam method entrap drug within the porous structure of foams and thus display a lattice diffusion mechanism. Theoretically, these two diffusion mechanisms can be identified by their activation energies for diffusion. With varying in vitro temperature, the activation energies were calculated from plots of ln (DIT) vs T-1 and in D vs T-1, where D is the diffusivity and T is the in vitro temperature in K. According to the results, we concluded that the INH from the dry-mixed matrices diffused through the drug channels filled with the medium, while the INH from the foam matrices diffused through the polymer lattice.

Antitubercular Agents↗

PubMed1995

Neurotrophin-4/5, brain-derived neurotrophic factor, and neurotrophin-3 promote survival of cultured vestibular ganglion neurons and protect them against neurotoxicity of ototoxins.

The ability of neurotrophin-4/5 (NT-4/5), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) to promote survival of postnatal rat vestibular ganglion neurons (VGNs) was examined in dissociated cell cultures. Of the four neurotrophins, NT-4/5 and BDNF were equally effective but more potent than NT-3 in promoting the survival of VGNs. In contrast, NGF showed no detectable effects. As expected, TrkB-IgG (a fusion protein of extracellular domain of TrkB and Fc domain of human immunoglobulin G) specifically inhibited the survival-promoting, effects by NT-4/5 or BDNF and TrkC-IgG fusion protein completely blocked that of NT-3. Immunohistochemistry with TrkB, TrkA, and p75 antisera revealed that VGNs made TrkB and p75 proteins, but not TrkA protein. Ototoxic therapeutic drugs such as cisplatin and gentamicin often induce degeneration of hair cells and ganglion neurons in both auditory and vestibular systems that leads to impairment of hearing and balance. When cisplatin and gentamicin were added to the dissociated VGN culture in which the hair cells were absent, additional cell death of VGNs was induced, suggesting that the two ototoxins may have a direct neurotoxic effect on ganglion neurons in addition to their known toxicity on hair cells. However, if the cultures were co-treated with neurotrophins, NT-4/5, BDNF, and NT-3, but not NGF, prevented or reduced the neurotoxicity of the two ototoxins. Thus, the three neurotrophins are survival factors for VGNs and are implicated in the therapeutic prevention of VGN loss caused by injury and ototoxins.

Animals↗

PubMed1995

Voltage-activated ionic currents in differentiating rat cerebellar granule neurons cultured from the external germinal layer.

The electrical properties of the precursor cells of the external germinal layer of rat cerebellum were assessed during their differentiation in control medium (Dulbecco's modified Eagle's medium) supplemented or not with either basic fibroblast growth factor (bFGF) or 25 mM potassium chloride (KCl). Resting potential was shown to be -10 mV in all three conditions 3 hours after plating [days in vitro (DIV)0]. By DIV 5, it reached -63 mV for cells cultured in 25 mM KCl but only -28 mV in control and bFGF media. The main voltage-sensitive ionic current measured at DIV 0 under all conditions was a composite IK consisting in a sustained K+ current blocked by tetraethylammonium (IK(TEA)), plus a rapidly activating and inactivating TEA-insensitive IK(A). Both currents increased with time in all conditions, but after 5 days IK(A) became dominant in terms of density. IK(TEA) is likely an IK(Ca), since it was blocked by 67% in 1 mM TEA. On DIV 0, INa and ICa were absent or small in amplitude. By DIV 3, 80% of the cells had currents able to generate a spike. Interestingly, ICa mean amplitude and current density measured at -10 mV in control condition on DIV 1 was significantly larger than those recorded in bFGF and 25 mM KCl. The order of appearance of the ionic currents, IK, ICa, and INa, leads directly to fast spike activity allowing for poor calcium entry. Firing rate likely depends on IK(A), which increased during the first 6 days of development but could be differentially regulated by bFGF.

Animals↗

PubMed1995

Clinical investigation of medical devices.

Clinical investigations are required for implantable and invasive devices in Class IIa or IIb, and all Class III devices. This article describes how to successfully perform a clinical trial according to the standards set in EN 540, Clinical Investigation of Medical Devices for Human Subjects.

Clinical Trials as Topic↗

PubMed1995

Neurotrophin-4/5 enhances survival of cultured spiral ganglion neurons and protects them from cisplatin neurotoxicity.

Destruction of spiral ganglion neurons (SGNs) induced by injury and toxins is one of the major causes for hearing loss. Here we report that neurotrophin-4/5 (NT-4/5), a member of the nerve growth factor family, promoted survival of postnatal rat SGNs up to threefold in dissociated cell cultures. The survival-promoting potency of NT-4/5 was equivalent to that of BDNF and stronger than that of NT-3. In contrast, NGF showed no detectable effects. Immunohistochemistry, with TrkB and TrkA antisera, revealed that these neurons produced TrkB protein, the functional receptor for NT-4/5 and BDNF, but not TrkA protein, the high-affinity receptor for NGF. The survival-promoting activity of NT-4/5 was completely inhibited by TrkB-IgG fusion protein. These results suggest that NT-4/5 is a specific survival factor for SGNs. In addition, NT-4/5 protected the SGNs from neurotoxic effects of the anti-cancer drug, cisplatin. Thus, NT-4/5 may have therapeutic value in preventing hearing impairment caused by damage to primary auditory afferent neurons.

Animals↗