Dennis E. Coyle

Partial peripheral nerve injury leads to activation of astroglia and microglia which parallels the development of allodynic behavior

Three different cell markers were studied in rats to note changes in the immunoreactivity (IR) in the lumbar spinal cord 1 to 84 days following partial sciatic nerve ligation (PSNL). Alteration in average IR were studied for complement receptor C3bi (OX42; microglia), major histocompatibility complex II (OX6; microglia), and glia fibrillary acidic protein (GFAP; astroglia). Thirty‐four female rats underwent ligation of approximately ½ of the sciatic nerve (PSNL). This injury resulted in the development of mechanical allodynia, which was quantitated by the measurement of foot withdrawal threshold to the application of Von Frey filaments. Ipsilateral increase in IR of OX42 and GFAP was observed to occur within 2 days, maximized by day 14, and did not return to contralateral spinal gray matter IR levels by day 84 (time period of study). Increases in OX42 IR and GFAP IR were observed within the spinal segments innervated by the sciatic nerve. GFAP IR was not expressed in all lumbar segments. OX42 staining with the upper portion of the dorsal horn was found to localize within the areas of deafferentation demonstrated by loss of thiamine monophosphatase activity within the substantia gelatinosa. OX6 IR was only seen in a few cells within the ipsilateral gray matter, indicating that microglia did not become phagocytic. Both GFAP IR and OX42 IR were found to linearly correlate with allodynic behavior with OX42 IR being more statistically significant. Correlation of OX42 IR in only the upper portion of the dorsal horn (not including the neck) resulted in an even a greater level of significance. These findings demonstrate that microglia and astroglia are activated following PSNL and that their increase in IR correlates with the development of allodynic behavior. GLIA 23:75–83, 1998.

Merkel Cells Transduce and Encode Tactile Stimuli to Drive Aβ-Afferent Impulses

Sensory systems for detecting tactile stimuli have evolved from touch-sensing nerves in invertebrates to complicated tactile end organs in mammals. Merkel discs are tactile end organs consisting of Merkel cells and Aβ-afferent nerve endings and are localized in fingertips, whisker hair follicles, and other touch-sensitive spots. Merkel discs transduce touch into slowly adapting impulses to enable tactile discrimination, but their transduction and encoding mechanisms remain unknown. Using rat whisker hair follicles, we show that Merkel cells rather than Aβ-afferent nerve endings are primary sites of tactile transduction and identify the Piezo2 ion channel as the Merkel cell mechanical transducer. Piezo2 transduces tactile stimuli into Ca(2+)-action potentials in Merkel cells, which drive Aβ-afferent nerve endings to fire slowly adapting impulses. We further demonstrate that Piezo2 and Ca(2+)-action potentials in Merkel cells are required for behavioral tactile responses. Our findings provide insights into how tactile end-organs function and have clinical implications for tactile dysfunctions.

Cardiovascular toxicity of local anesthetics: an alternative hypothesis.

The current study examined the possibility that high local anesthetic concentrations within the central nervous system could contribute to the toxic cardiovascular effects observed clinically after an accidental intravenous injection. Equal numbers of molecules of lidocaine (1.6 μl of a 2% solution) and bupivacaine (1.0 μl of a 4% solution) were injected at three vasomotor and cardioactive areas in the rat medulla by means of a 28-gauge hypodermic needle and a micro-syringe. These areas were the C1 region, the intermedialateral column (IML), and the nucleus tractus solitarius (NTS). Both lidocaine and bupivacaine at C1 significantly decreased mean arterial pressure and, at IML, resulted in significant bradycardia and hypotension. At NTS, both lidocaine and bupivacaine caused significant bradycardia and hypotension, which were accompanied by ventricular arrhythmias in 55% of the animals. In all animals in whom ventricular arrhythmias were associated with lidocaine, the arrhythmias spontaneously reverted to normal sinus rhythm. In 50% of animals developing ventricular arrhythmias after bupivacaine, the arrhythmias resulted in death. Using an equal number of molecules of lidocaine and bupivacaine, all three regions studied demonstrated that bupivacaine was 2–4 times more potent than lidocaine in producing cardiovascular effects. These data demonstrate that direct application of local anesthetics within the medullary region of the central nervous system can result in hypotension, bradycardia, and ventricular arrhythmias similar to what may be seen in humans after accidental intravenous injections of local anesthetics. Moreover, the sites and mechanisms of action appear to be identical for bupivacaine and lidocaine. Thus bupivacaine does not appear to be an aberrant local anesthetic, as some have suggested, but rather produces more profound effects related to its potency and physico-chemical properties.

Female rats are more susceptible to the development of neuropathic pain using the partial sciatic nerve ligation (PSNL) model

A comparison study was conducted to determine if a gender difference could be detected using an animal model for causalgia. The sciatic nerve was tightly ligated so that 1/3 to 1/2 of the nerve thickness was trapped by the ligature, just distal to the point at which the posterior biceps semitendinosus nerve branches off the common sciatic nerve. By measuring paw withdrawal from innocuous stimulation with Von Frey filaments, the percent of rats displaying average mechanical sensitivity of the injured paw that was significantly elevated compared to sham or unoperated control animals (days 22-24) was 28.6% for the male group versus 63.6% for the female group. Our animals did not display a consistent response in withdrawal latency to heat applied to the plantar surface of the root (hyperpathia). The data suggests that female rats are significantly more susceptible to developing neuropathic pain than male rats using this experimental model for causalgia.

The Chemokine CXCL1/Growth Related Oncogene Increases Sodium Currents and Neuronal Excitability in Small Diameter Sensory Neurons

BackgroundAltered Na+ channel expression, enhanced excitability, and spontaneous activity occur in nerve-injury and inflammatory models of pathological pain, through poorly understood mechanisms. The cytokine GRO/KC (growth related oncogene; CXCL1) shows strong, rapid upregulation in dorsal root ganglion in both nerve injury and inflammatory models. Neurons and glia express its receptor (CXCR2). CXCL1 has well-known effects on immune cells, but little is known about its direct effects on neurons.ResultsWe report that GRO/KC incubation (1.5 nM, overnight) caused marked upregulation of Na+ currents in acutely isolated small diameter rat (adult) sensory neurons in vitro. In both IB4-positive and IB4-negative sensory neurons, TTX-resistant and TTX-sensitive currents increased 2- to 4 fold, without altered voltage dependence or kinetic changes. These effects required long exposures, and were completely blocked by co-incubation with protein synthesis inhibitor cycloheximide. Amplification of cDNA from the neuronal cultures showed that 3 Na channel isoforms were predominant both before and after GRO/KC treatment (Nav 1.1, 1.7, and 1.8). TTX-sensitive isoforms 1.1 and 1.7 significantly increased 2 – 3 fold after GRO/KC incubation, while 1.8 showed a trend towards increased expression. Current clamp experiments showed that GRO/KC caused a marked increase in excitability, including resting potential depolarization, decreased rheobase, and lower action potential threshold. Neurons acquired a striking ability to fire repetitively; IB4-positive cells also showed marked broadening of action potentials. Immunohistochemical labelling confirmed that the CXCR2 receptor was present in most neurons both in dissociated cells and in DRG sections, as previously shown for neurons in the CNS.ConclusionMany studies on the role of chemokines in pain conditions have focused on their rapid and indirect effects on neurons, via release of inflammatory mediators from immune and glial cells. Our study suggests that GRO/KC may also have important pro-nociceptive effects via its direct actions on sensory neurons, and may induce long-term changes that involve protein synthesis.

Alpha1‐acid glycoprotein and albumin in human serum bupivacaine binding

Bupivacaine protein binding was studied with the use of human serum, isolated human serum albumin, and isolated α1‐acid glycoprotein. The effect of lactic acid on bupivacaine binding was also studied. Bupivacaine protein binding in serum is best characterized by a model described by two classes of binding sites and that in α1‐acid glycoprotein or albumin is best characterized by a model described by one class of binding sites for each protein. Albumin binding closely correlated with the data obtained for the low‐affinity, high‐capacity binding site in serum, while α1‐acid glycoprotein data closely correlated with the data obtained for the high‐affinity, low‐capacity site in serum. A reduction in pH resulted in a significant reduction in the affinity for the high‐affinity, low‐capacity site in serum. No other binding parameters were affected. These data were in excellent agreement with results of the isolated protein studies. Our data demonstrate that acidosis results in significant increases in free bupivacaine concentrations only at relatively low total bupivacaine concentrations (<7 µg/ml) and that distribution characteristics for bupivacaine are essentially unchanged over a wide concentration range.

Intact female rats are more susceptible to the development of tactile allodynia than ovariectomized female rats following partial sciatic nerve ligation (PSNL)

As previously reported, this laboratory has determined that female rats are more prone to develop tactile allodynia, an indicator of neuropathic pain, than male rats using the partial sciatic nerve ligation (PSNL) model. In order to further characterize this gender difference, the role of ovarian hormones in predisposing female rats to the development of tactile allodynia was investigated. In a double blind randomized trial, 12 intact and 12 ovariectomized (ovx) female rats underwent PSNL of the right rear leg. Sham 1 operated (exposure of the nerve without ligation; 2 intact, 2 ovx) and sham 2 operated (incision through skin only; 1 intact, 2 ovx) controls were also included in the study groups. Animals were evaluated on 3 consecutive days for withdrawal from touch with von Frey filaments on post-injury days 13-15, 20-22 and 27-29. A significant difference was observed at post-injury days 20-22 and 27-29 between the two groups. The intact group resulted in 12/12 animals that were statistically more sensitive than sham groups, compared to 6/12 for the ovx group. At post-injury days 13-15 no statistical difference was seen between the two groups (8/12 for intact versus 10/12 for ovx). This study indicates that ovarian hormones, via some unknown mechanism, predispose female rats to develop tactile allodynia following injury.

Absolute Bioavailability of Hydromorphone After Peroral and Rectal Administration in Humans: Saliva/Plasma Ratio and Clinical Effects

The absolute bioavailability of hydromorphone was investigated in eight healthy male subjects by a cross‐over design (with washout period of two weeks) after intravenous (2 mgj, peroral (4 mg) and rectal (3 mg) administration of hydromorphone. The use of saliva hydromorphone concentrations as a noninvasive technique in pharmacokinetic evaluation was investigated, and the clinical effects after the three routes of administration were determined. Hydromorphone has an absolute bioavailability of 51.35 ± 29.29% and 36.33 ± 29.60% after peroral and rectal administration, respectively. More side effects were observed after intravenous administration of hydromorphone than after rectal or perorai dosing. The saliva sampling for the hydromorphone concentration was found to be a useful noninvasive technique for the estimation of the elimination half‐life of hydromorphone.

Microarray Analysis of Rat Sensory Ganglia after Local Inflammation Implicates Novel Cytokines in Pain

Inflammation plays a role in neuropathic pain conditions as well as in pain induced solely by an inflammatory stimulus. Robust mechanical hyperalgesia and allodynia can be induced by locally inflaming the L5 dorsal root ganglion (DRG) in rat. This model allows investigation of the contribution of inflammation per se to chronic pain conditions. Most previous microarray studies of DRG gene expression have investigated neuropathic pain models. To examine the role of inflammation, we used microarray methods to examine gene expression 3 days after local inflammation of the L5 DRG in rat. We observed significant regulation in a large number of genes (23% of observed transcripts), and examined 221 (3%) with a fold-change of 1.5-fold or more in more detail. Immune-related genes were the largest category in this group and included members of the complement system as well as several pro-inflammatory cytokines. However, these upregulated cytokines had no prior links to peripheral pain in the literature other than through microarray studies, though most had previously described roles in CNS (especially neuroinflammatory conditions) as well as in immune responses. To confirm an association to pain, qPCR studies examined these cytokines at a later time (day 14), as well as in two different versions of the spinal nerve ligation pain model including a version without any foreign immunogenic material (suture). Cxcl11, Cxcl13, and Cxcl14 were found to be significantly upregulated in all these conditions, while Cxcl9, Cxcl10, and Cxcl16 were upregulated in at least two of these conditions.

Bupivacaine protein binding in the term parturient: Effects of lactic acidosis

The effects of lactic acidosis on bupivacaine serum protein binding was studied in a group of term parturients and a group of nonpregnant female control subjects. Groups were matched in age and health. Distribution characteristics of bupivacaine in pregnancy were determined. Bupivacaine protein binding was best characterized by the model for two classes of binding sites in all studies. The parturients exhibited a lower capacity for the high‐affinity, low‐capacity (α1‐acid glycoprotein) site and higher affinity for the low‐affinity, high‐capacity (albumin) site. Lactic acidosis decreased the affinity constant for the high‐affinity, low‐capacity site in the control group but did not change binding characteristics in the parturients. Free concentration of bupivacaine (Cu) was elevated at low total bupivacaine concentrations (Ct) (<10 µg/ml). No differences in Cu were detected at concentrations in the cardiotoxic range (>20 µg/ml). The Cu values predicted by the estimated binding parameters from in vitro experiments were compared with actual Cu measured in nine parturients at delivery; they correlated significantly (r = 0.94). Distribution changes for bupivacaine in the parturients were consistent with known physiologic changes in body composition associated with pregnancy. Alterations that occur in serum protein binding during pregnancy should not result in increased risk of central nervous system or cardiovascular system toxicity since these alterations do not increase free tissue concentration.