Chemical responsiveness and histochemical phenotype of electrophysiologically classified cells of the adult rat dorsal root ganglion.

Whole cell patch recordings were obtained from medium diameter (35-45 microm) dorsal root ganglion neurons. Using electrophysiological parameters, we were able to subclassify acutely dissociated dorsal root ganglion cells into three uniform (types 5, 6 and 9) and one mixed class (type 8) of neurons. All subtypes (types 5, 6, 8 and 9) had broad action potentials (7.0+/-0.2, 5.2+/-0.4, 7.3+/-0.5 and 6.0+/-0.4 ms) and exceptionally long afterhyperpolarizations (112+/-9, 178+/-19, 124+/-31 and 204+/-33 ms). Long afterhyperpolarizations have been linked to mechanically insensitive (silent) nociceptors by other laboratories [Djouhri et al., J. Physiol. 513 (1998) 857-872]. Chemosensitivity varied among cell classes. Cell types 5, 8 and 9 were capsaicin sensitive (45+/-13, 87+/-30 and 28+/-13 pA/pF; 5 microM) groups, while the type 6 cell was capsaicin insensitive. All cell types expressed ASIC-like (acid sensing ion channel) amiloride sensitive, proton-activated currents with a threshold of pH 6.8 and a peak near pH 5.0. All medium sized cells were sensitive to ATP (50 microM) and exhibited the 'mixed' form of ATP-gated current [Burgard et al., J. Neurophysiol. 82 (1999) 1590-1598; Grubb and Evans, Eur. J. Neurosci. 11 (1999) 149-154]. Immunohistochemistry performed on individual cells indicated the expression of both P2X(1) and P2X(3) subunits. Electrophysiologically defined classes were histochemically uniform. All types were examined for the presence of substance P (SP), calcitonin gene related peptide (CGRP) and binding of isolectin B4 (IB4). All subtypes expressed CGRP immunoreactivity. Types 5 and 8 co-expressed SP and CGRP immunoreactivity and also bound IB4. Subtypes 6 and 9 were positive for neurofilament m. It is likely that these cells represent major classes of myelinated and unmyelinated peptide expressing nociceptors.

Myotoxicity studies of injectable biodegradable in-situ forming drug delivery systems.

The objective of the study was to investigate the potential in-vitro and in-vivo myotoxicity of different in-situ forming biodegradable drug delivery systems, namely in-situ Microparticle (ISM) systems and polymer solutions (in-situ implant systems). The acute myotoxicity was evaluated in-vitro using the isolated rodent skeletal muscle model by measuring the cumulative creatine kinase (CK) efflux. For the in-vivo study, following intramuscular injection (i.m.) into male Sprague Dawley rats, the area under the plasma CK-curve was used to evaluate muscle damage. The formulations included ISM-systems [a poly (lactide)-solvent phase dispersed into an external oil phase] and poly (lactide) solutions (in-situ implant systems). Phenytoin and normal saline served as positive and negative controls, respectively. Poly (lactide) in different solvents (in-situ implant systems) resulted in 14.4-24.3 times higher CK-values compared to normal saline, indicating a high myotoxic potential. With the ISM-system, the CK-release was significantly lower, decreased with a lower polymer phase: oil phase ratio, and approached the values of normal saline at a ratio of 1:4. Bupivacaine HCl- and Buserelin acetate- containing ISM-systems resulted in significantly lower CK-levels when compared to the corresponding drug formulation in normal saline. The in-vivo studies confirmed the in-vitro data and showed good muscle compatibility of the ISM-systems.

Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents.

We used a "current signature" method to subclassify acutely dissociated dorsal root ganglion (DRG) cells into nine subgroups. Cells subclassified by current signature had uniform properties. The type 1 cell had moderate capsaicin sensitivity (25.9 pA/pF), powerful, slowly desensitizing (tau = 2,300 ms), ATP-activated current (13.3 pA/pF), and small nondesensitizing responses to acidic solutions (5.6 pA/pF). Type 1 cells expressed calcitonin gene-related peptide immunoreactivity (CGRP-IR), manifested a wide action potential (7.3 ms), long duration afterhyperpolarization (57.0 ms), and were IB4 positive. The type 2 cell exhibited large capsaicin activated currents (134.9 pA/pF) but weak nondesensitizing responses to protons (15.3 pA/pF). Currents activated by ATP and alphabeta-m-ATP (51.7 and 44.6 pA/pF, respectively) had fast desensitization kinetics (tau = 214 ms) that were distinct from all other cell types. Type 2 cells were IB4 positive but did not contain either substance P (SP) or CGRP-IR. Similar to capsaicin-sensitive nociceptors in vivo, the afterhyperpolarization of the type 2 cell was prolonged (54.7 ms). The type 3 cell expressed, amiloride-sensitive, rapidly desensitizing (tau = 683 ms) proton-activated currents (127.0 pA/pF), and was insensitive to ATP or capsaicin. The type 3 cell was IB4 negative and contained neither CGRP nor SP-IR. The afterhyperpolarization (17.5 ms) suggested nonnociceptive function. The type 4 cell had powerful ATP-activated currents (17.4 pA/pF) with slow desensitization kinetics (tau = 2, 813 ms). The afterhyperpolarization was prolonged (46.5 ms), suggesting that this cell type might belong to a capsaicin-insensitive nociceptor population. The type 4 cell did not contain peptides. The type 7 cell manifested amiloride-sensitive, proton-activated currents (45.8 pA/pF) with very fast desensitization kinetics (tau = 255 ms) and was further distinct from the type 3 cell by virtue of a nondesensitizing amiloride-insensitive component (6.0 pA/pF). Capsaicin and ATP sensitivity were relatively weak (4.3 and 2.9 pA/pF, respectively). Type 7 cells were IB4 positive and contained both SP and CGRP-IR. They exhibited an exceptionally long afterhyperpolarization (110 ms) that was suggestive of a silent (mechanically insensitive) nociceptor. We concluded that presorting of DRG cells by current signatures separated them into internally homogenous subpopulations that were distinct from other subclassified cell types.

In situ gel formulations for gene delivery: release and myotoxicity studies.

The in vitro release of plasmid DNA and salmon sperm DNA from in situ gel formulations was investigated. Two in situ gel systems were studied: (a) an interpolymeric complex (IPC) of water-soluble polymers polymethacrylic acid (PMA) and polyethylene glycol (PEG) and (b) a hydroxypropylmethylcellulose-carbopol system (H:C). Two-way analysis of variance with replication demonstrated that both gel composition and medium pH influenced significantly the release of plasmid DNA from in situ gel formulations. When the release of both types of DNA was compared, higher release was observed for plasmid DNA compared to genomic salmon sperm DNA. Conformational analysis of the released plasmid DNA showed that DNA was released without degradation, but with remarkable conversion from supercoiled (SC) to open circular (OC). In addition, the tested in situ gel systems demonstrated protection from DNAse I degradation. The myotoxicity of the injectable gelling solutions was assessed by the cumulative release of creatine kinase (CK) over 120 min from the isolated rodent extensor digitorum longus (EDL) muscle. A higher level of cumulative CK was observed for IPC when compared to H:C (2:1). These results demonstrate that the in situ gelling systems can be considered as a valuable injectable controlled-delivery system for pDNA in their role to provide protection from DNAse degradation.

Assessment of the myotoxicity of pharmaceutical buffers using an in vitro muscle model: effect of pH, capacity, tonicity, and buffer type.

The purpose of the present study was to investigate the myotoxicity of three buffers containing carboxylic acid groups (i.e., acetate, succinate, and citrate) as a function of their pH, capacity, and tonicity. The myotoxicity of these buffers in the range of pH 2-6 and 0.001-0.1 M buffer capacity was assessed using cumulative creatine kinase (CK) release from an isolated rodent muscle model following injection. Phenytoin and 0.9% NaCl injection were used as positive and negative controls, respectively. Buffer solutions were prepared. A lower pH and higher buffer capacity was linked to increased myotoxicity for the acetate buffers. However, for succinate and citrate buffers, pH appeared to influence the extent of myotoxicity, whereas buffer capacity did not seem to have an effect. When either NaCl or trehalose was used as a tonicity-adjusting agent at pH 6, isotonic 0.01 M buffer solutions dramatically lowered the cumulative CK release compared to those that were not isotonic. Isotonic succinate buffers displayed the lowest myotoxicity, whereas citrate buffers displayed the highest values. Citrate buffers containing three carboxylic acid groups showed higher myotoxicity than succinate buffers and acetate buffers at 0.001 and 0.01 M buffer capacities, whereas acetate buffer produced higher cumulative CK release than citrate and succinate buffers at 0.1 M buffer capacity. The myotoxicity of pharmaceutical buffers containing carboxylic acid groups appears to be directly affected by lowering the pH of the solution.