Recent Publications by CFE Educators

The method of transneuronal retrograde transport of the Bartha strain of the swine alpha-herpes virus, pseudorabies virus, was used to identify putative interneurons presynaptic to motoneurons that supply a tail-flick muscle in the rat. We also investigated whether these interneurons also contribute to ascending somatosensory pathways. Two to five days after injection of pseudorabies virus into the left abductor caudae dorsalis muscle, and cholera toxin B into the right somatosensory thalamus and midbrain, rats were perfused and spinal cord sections processed immunohistochemically in a two-step procedure to stain cholera toxin B-immunoreactive cells black and pseudorabies virus-immunoreactive cells brown. At short (two-day) survivals, the first spinal neurons to be pseudorabies virus-immunoreactive were in the ipsilateral abductor caudae dorsalis motoneuron pool (S3-S4) and intermediolateral cell column (T12-L2), with a few (0 to five/section) bilaterally in the intermediate zone and around the central canal (all lumbosacral levels). With longer (three- to four-day) survival, more cells were noted (20-50/section) bilaterally (ipsilateral preponderance) in the dorsal and ventral horns of the lumbosacral cord. Many were in lamina I (marginal layer), while few were in lamina II (substantia gelatinosa). At four- and five-day survivals, the numbers of cells increased (20 to 100/section) bilaterally and now included lamina II. The fact that unilateral rhizotomy at L4-Co1 failed to change the distribution of spinal pseudorabies virus labeling suggests that the labeling was due to retrograde transport via the ventral root. In support, bilateral removal of the lumbar sympathetic ganglia, which receive their preganglionic innervation through the ventral root, reduced pseudorabies virus immunoreactivity throughout the thoracic and rostral lumbar spinal cord. These data indicate that there are (i) direct projections from intermediate and dorsal horn cells to abductor caudae dorsalis motoneurons, and (ii) disynaptic connections from dorsal horn (possibly including lamina II) cells to more ventral last-order interneurons. We also suggest that some lamina II cells are presynaptic to lamina I cells that project directly to abductor caudae dorsalis motoneurons. We observed cholera toxin B-immunoreactive cells (five to 20/section) in the expected locations (contralateral lamina I, deep dorsal horn and intermediate zone; lateral spinal nucleus bilaterally). Double-labeled (i.e. pseudorabies virus- and cholera toxin B-immunoreactive) neurons were only occasionally seen in the lateral spinal nucleus and were absent in the spinal gray matter, indicating that segmental interneurons do not collateralize in long ascending sensory pathways to the midbrain and somatosensory thalamus.

Injection of dilute formalin into the hindpaw produces brief (phase 1) and persistent (phase 2) nociceptive responses in the rat. We recently reported that ongoing peripheral nerve input is required for the expression of behavioral and cardiovascular responses during phase 2. Here we evaluated the contribution of central and peripheral sensitization mechanisms, generated during phase 1, to the magnitude and temporal profile of phase 2. During phase 1, we administered analgesic doses of an ultrashort-acting opioid, remifentanil (i.v. administration from 0-5 min after 5.0% formalin injection), or anesthetic concentrations of halothane (2.1%). Inhibition of phase 1 did not reduce the magnitude of flinching and cardiovascular responses during phase 2, but it did delay their onset and/or termination. Longer remifentanil infusions (0-15 or 0-30 min) produced even longer delays (up to 30 min) in the onset and termination of flinching during phase 2; however, when remifentanil was administered during the early part of phase 2 (15-30 or 15-45 min), it did not prolong the time to termination of phase 2. Continuous infusion (10 mg/kg/hr i.v.) of a peripherally acting opiate antagonist, naloxone methiodide, did not reduce the antinociception produced by remifentanil during phase 1 but almost completely reversed the delay in the onset and termination of phase 2. We conclude that central sensitization mechanisms during phase 1 do not influence the magnitude of phase 2. We also hypothesize that remifentanil interacts with peripheral opioid receptors to impede the formalin-evoked synthesis and/or release of proinflammatory compounds during phase 1 and thus delay phase 2.

In this study, we monitored Fos-like immunoreactivity in the sacral spinal cord to identify neurons that are likely to contribute to the autonomic manifestations of opioid antagonist-precipitated withdrawal in morphine-tolerant rats. Injection of systemic antagonist increased the Fos-like immunoreactivity throughout the first sacral segment, particularly in laminae I/II, X, and in the sacral parasympathetic nucleus (SPN). Selective peripheral withdrawal, with a hydrophilic antagonist that does not cross the blood-brain barrier (BBB), induced diarrhea, but no other withdrawal signs were evident. Compared to rats that withdrew systemically, peripherally withdrawal evoked significantly less Fos-like immunoreactivity in laminae V/VI, X and the SPN. By contrast, selective spinal withdrawal, by intrathecal injection of an opioid antagonist that does not cross the BBB, provoked hyperactivity of the hindlimbs and tail, but no diarrhea. These animals demonstrated significantly increased Fos-like immunoreactivity in laminae I/II, V/VI, the SPN, and the ventral horn compared to rats that withdrew systemically. Animals treated neonatally with capsaicin, to eliminate C-fiber input, demonstrated withdrawal behavior similar to intact withdrawing rats, except that the capsaicin-pretreated rats had significantly greater weight loss. However, this group had less Fos-like immunoreactivity in laminae V/VI, X and SPN compared to the intact withdrawing rats. These data suggest that withdrawal from morphine evokes hyperactivity of sacral neurons, particularly those involved in regions that process nociceptive and autonomic information. Peripheral withdrawal is sufficient to induce diarrhea, but it does not fully explain the associated weight loss. Unmyelinated primary afferents may contribute a tonic peripheral inhibition of circuits that regulate gut motility and intestinal fluid transport. Taken together, these data suggest that chronic exposure to opioids induces a latent sensitization in sacral cord neurons that can be manifested as neuronal hyperactivity during withdrawal; this mechanism may underlie withdrawal-induced hyperalgesia and gut hypermotility.

Injection of formalin in the rat hindpaw produces two phases of nociceptive behavior. Although it is generally agreed that the first phase results from direct chemical activation of nociceptive primary afferent fibers, the factors that contribute to the second phase are not established. In the present study, we monitored the expression of the c-fos protein to evaluate whether the pattern of activity of dorsal horn neurons differs as a result of ongoing afferent activity during the two phases. To selectively block the first or second phase, we respectively used remifentanil, a potent and short acting opiate agonist, and QX-314, a quaternary derivative of lidocaine, which does not cross the blood brain barrier. We also evaluated the effect of eliminating nociceptive behavior in both phases using both remifentanil and lidocaine or a combination of local anesthetics, bupivicaine and quaternary lidocaine. In all groups, formalin (5%, 50 microliters) was injected subcutaneously into the plantar surface of the hindpaw. To assess the nociceptive behavior produced by formalin, we monitored the number of flinches. Injection of remifentanil during the first phase completely blocked the first phase formalin-evoked nociceptive behavior, and had no effect on the second phase. Injection of lidocaine during the interphase completely blocked second phase nociceptive behavior. As expected, when remifentanil was administered during the first phase and lidocaine during the second phase, all formalin-evoked nociceptive behavior was blocked. The same was true for rats that received injections of bupivicaine and lidocaine during phases 1 and 2, respectively. In laminae I-II of the L4-L5 segment, the magnitude of the decrease in Fos expression was comparable for remifentanil (26.5%) and lidocaine (27.3%); the decrease was greater when both remifentanil and lidocaine were administered (50.5%), and even greater when bupivicaine and lidocaine were used (74.2%). In laminae V-VI, remifentanil, by itself, decreased c-fos expression by 39.4%; for lidocaine alone, the decrease was 58.4%. We did not observe further significant decreases when both remifentanil and lidocaine, or bupivacaine and lidocaine were injected (69.7% and 74.6%, respectively). Our results not only provide strong evidence that activity during the second phase is necessary for maintaining the maximal expression of c-fos in the spinal cord, but also reveal significant regional differences in the central patterns of activity generated during the two phases. These results also confirm our previous reports that c-fos expression is not eliminated when the behavioral manifestation of the noxious stimulus is completely blocked.

The opioid antagonist, naloxone, produces equivocal effects on the magnitude of nociceptive responses in several animal models of persistent pain, including the formalin test. Hindpaw injection of dilute formalin produces not only inflammation but also phasic (Phase 1) and persistent (Phase 2) behavioral and cardiovascular nociceptive responses in the rat. To test the hypothesis that endogenous opioid systems contribute to the magnitude of responses to intraplantar formalin injection, we evaluated the effects of continuous naloxone administration (0.01-100 mg/kg per h, i.v.) on formalin-evoked hindpaw inflammation, on behavioral indices of pain, flinching and licking pain behavior, and on changes in mean arterial pressure and heart rate. We report that naloxone, at doses less than 100 mg/kg per h, did not change any formalin-evoked response. Although the 100 mg/kg per h dose significantly decreased these responses, it also produced muscle rigidity and profound bradycardia. We conclude that endogenous opioids do not significantly modulate the nociceptive processing induced by subcutaneous formalin.

The 39 kDa receptor-associated protein (RAP) is a receptor antagonist that interacts with several members of the low density lipoprotein (LDL) receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. Our recent studies have demonstrated that RAP is an endoplasmic reticulum (ER) resident protein and an intracellular chaperone for the LDL receptor-related protein (LRP). The HNEL sequence at the carboxyl terminus of RAP represents a novel ER retention signal that shares homology with the well-characterized KDEL signal. In the present study, using immunoelectron microscopy we demonstrate that cells stably transfected with human growth hormone (GH) tagged with either KDEL (GH + KDEL) or HNEL (GH + HNEL) signals exhibit ER and cis-Golgi localization typical of ER-retained proteins. Overexpression of not only GH + HNEL but also GH + KDEL cDNA in transfected cells results in saturation of ER retention receptors and secretion of endogenous RAP indicating that the two signals interact with the same ER retention receptor(s). The role of RAP in the maturation of LRP is further supported by the observation that functional LRP is reduced about 60% as a result of decreased intracellular RAP. Pulse-chase labeling and immunolocalization studies of ERD2.1 and ERD2.2 proteins in transfected cells demonstrate a long half-life and Golgi localization for both receptors. Finally, overexpression of either ERD2.1 or ERD2.2 proteins significantly increases the capacity of cells to retain both KDEL and HNEL-containing proteins. Taken together, our results thus demonstrate that ERD2 proteins are capable of retaining the novel ER retention signal associated with RAP.

While ambulatory care education typically does not provide much direct instruction, supervision, or feedback, experiential learning occurs. Using experiential learning theory, the authors describe how this process of learning works. the process is characterized by a cycle of having a concrete experience (e.g., an encounter with a patient), reflecting on that experience as it unfolds, formulating conceptualizations and generalizations from the experience, and testing those generalizations and concepts in other situations. With this model in mind, the authors make four recommendations for improving ambulatory care education for both medical students and residents: (1) plan for experiences in carefully selected ambulatory care settings; (2) facilitate reflective observation; (3) encourage conceptual thinking and inquiry; and (4) promote feedback and testing of insights from experiences. The authors discuss the rationale behind each recommendation and offer guidelines for how each might be implemented.