A developmental assessment of clinical reasoning in preclinical medical education.

BACKGROUND: Clinical reasoning is an essential skill to be learned during medical education. A developmental framework for the assessment and measurement of this skill has not yet been described in the literature. OBJECTIVE: The authors describe the creation and pilot implementation of a rubric designed to assess the development of clinical reasoning skills in pre-clinical medical education. DESIGN: The multi-disciplinary course team used Backwards Design to develop course goals, objectives, and assessment for a new Clinical Reasoning Course. The team focused on behaviors that students were expected to demonstrate, identifying each as a 'desired result' element and aligning these with three levels of performance: emerging, acquiring, and mastering. RESULTS: The first draft of the rubric was reviewed and piloted by faculty using sample student entries; this provided feedback on ease of use and appropriateness. After the first semester, the course team evaluated whether the rubric distinguished between different levels of student performance in each competency. A systematic approach based on descriptive analysis of mid- and end of semester assessments of student performance revealed that from mid- to end-of-semester, over half the students received higher competency scores at semester end. CONCLUSION: The assessment rubric allowed students in the early stages of clinical reasoning development to understand their trajectory and provided faculty a framework from which to give meaningful feedback. The multi-disciplinary background of the course team supported a systematic and robust course and assessment design process. The authors strongly encourage other colleges to support the use of collaborative and multi-disciplinary course teams.

Bone enhancement in ultrasound using local spectrum variations for guiding percutaneous scaphoid fracture fixation procedures.

PURPOSE: The scaphoid bone is the most frequently fractured bone in the wrist. When fracture fixation is indicated, a screw is inserted into the bone either in an open surgical procedure or percutaneously under fluoroscopic guidance. Due to the complex geometry of the wrist, fracture fixation is a challenging task. Fluoroscopic guidance exposes both the patient and the physician to ionizing radiation. Ultrasound-based guidance has been suggested as a real-time, radiation-free alternative. The main challenge of using ultrasound is the difficulty in interpreting the images due to the low contrast and noisy nature of the data. METHODS: We propose a bone enhancement method that exploits local spectrum features of the ultrasound image. These features are utilized to design a set of quadrature band-pass filters and subsequently estimate the local phase symmetry, where high symmetry is expected at the bone locations. We incorporate the shadow information below the bone surfaces to further enhance the bone responses. The extracted bone surfaces are then used to register a statistical wrist model to ultrasound volumes, allowing the localization and interpretation of the scaphoid bone in the volumes. RESULTS: Feasibility experiments were performed using phantom and in vivo data. For phantoms, we obtain a surface distance error 1.08 mm and an angular deviation from the main axis of the scaphoid bone smaller than 5°, which are better compared to previously presented approaches. CONCLUSION: The results are promising for further development of a surgical guidance system to enable accurate anatomy localization for guiding percutaneous scaphoid fracture fixations.

Competing molecular interactions of aPKC isoforms regulate neuronal polarity.

Atypical protein kinase C (aPKC) isoforms ζ and λ interact with polarity complex protein Par3 and are evolutionarily conserved regulators of cell polarity. Prkcz encodes aPKC-ζ and PKM-ζ, a truncated, neuron-specific alternative transcript, and Prkcl encodes aPKC-λ. Here we show that, in embryonic hippocampal neurons, two aPKC isoforms, aPKC-λ and PKM-ζ, are expressed. The localization of these isoforms is spatially distinct in a polarized neuron. aPKC-λ, as well as Par3, localizes at the presumptive axon, whereas PKM-ζ and Par3 are distributed at non-axon-forming neurites. PKM-ζ competes with aPKC-λ for binding to Par3 and disrupts the aPKC-λ-Par3 complex. Silencing of PKM-ζ or overexpression of aPKC-λ in hippocampal neurons alters neuronal polarity, resulting in neurons with supernumerary axons. In contrast, the overexpression of PKM-ζ prevents axon specification. Our studies suggest a molecular model wherein mutually antagonistic intermolecular competition between aPKC isoforms directs the establishment of neuronal polarity.

A novel nicotinic mechanism underlies ß-amyloid-induced neuronal hyperexcitation.

There is a significantly elevated incidence of epilepsy in Alzheimer's disease (AD). Moreover, there is neural hyperexcitation/synchronization in transgenic mice expressing abnormal levels or forms of amyloid precursor protein and its presumed, etiopathogenic product, amyloid-ß1-42 (Aß). However, the underlying mechanisms of how Aß causes neuronal hyperexcitation remain unclear. Here, we report that exposure to pathologically relevant levels of Aß induces Aß form-dependent, concentration-dependent, and time-dependent neuronal hyperexcitation in primary cultures of mouse hippocampal neurons. Similarly, Aß exposure increases levels of nicotinic acetylcholine receptor (nAChR) α7 subunit protein on the cell surface and α7-nAChR function, but not α7 subunit mRNA, suggesting post-translational upregulation of functional α7-nAChRs. These effects are prevented upon coexposure to brefeldin A, an inhibitor of endoplasmic reticulum-to-Golgi protein transport, consistent with an effect on trafficking of α7 subunits and assembled α7-nAChRs to the cell surface. Aß exposure-induced α7-nAChR functional upregulation occurs before there is expression of neuronal hyperexcitation. Pharmacological inhibition using an α7-nAChR antagonist or genetic deletion of nAChR α7 subunits prevents induction and expression of neuronal hyperexcitation. Collectively, these results, confirmed in studies using slice cultures, indicate that functional activity and perhaps functional upregulation of α7-nAChRs are necessary for production of Aß-induced neuronal hyperexcitation and possibly AD pathogenesis. This novel mechanism involving α7-nAChRs in mediation of Aß effects provides potentially new therapeutic targets for treatment of AD.

Volume slicing of cone-beam computed tomography images for navigation of percutaneous scaphoid fixation.

PURPOSE: Percutaneous scaphoid fixation (PSF) is growing in popularity as a treatment option for non-displaced fractures. Success of this procedure demands high-precision screw placement, which can be difficult to achieve with standard 2D imaging. This study aimed to develop and test a system for computer-assisted navigation using volume slicing of 3D cone-beam computed tomography (CBCT). METHODS: The navigated technique involved a distinctive workflow in which a 3D CBCT imager was calibrated preoperatively, circumventing the need for intraoperative patient-based registration. Intraoperatively, a 3D CBCT image was acquired for both preoperative planning and direct navigation using volume-rendered slices. An in vitro study was conducted to compare the navigated approach to two conventional fluoroscopic methods for volar PSF. The surgical goal was to insert a guide wire to maximize both length and central placement. RESULTS: There was no significant difference in the mean central placement of guide wire, although the variance in central placement was significantly lower using VS navigation (P < 0.01). The lengths of the drill paths were significantly longer for the VS-navigated group compared with one 2D group (P < 0.1). Each navigated trial required only one drilling attempt and resulted in less radiation exposure than conventional C-arm (P < 0.01). CONCLUSIONS: Volume-sliced navigation achieved a more repeatable and reliable central pin placement, with fewer drilling attempts than conventional 2D techniques. Volume-sliced navigation had a higher number of drill paths within the optimal zone maximizing both length of the path and depth from the surface.

Calibration and use of intraoperative cone-beam computed tomography: an in-vitro study for wrist fracture.

The standard workflow in many image-guided procedures, preoperative imaging followed by intraoperative registration, can be a challenging process and is not readily adaptable to certain anatomical regions such as the wrist. In this study we present an alternative, consisting of a preoperative registration calibration and intraoperative navigation using 3D cone-beam CT. A custom calibration tool was developed to preoperatively register an optical tracking system to the imaging space of a digital angiographic C-arm. This preoperative registration was then applied to perform direct navigation using intraoperatively acquired images for the purposes of an in-vitro wrist fixation procedure. A validation study was performed to assess the stability of the registration and found that the mean registration error was approximately 0.3 mm. When compared to two conventional techniques, our navigated wrist repair achieved equal or better screw placement, with fewer drilling attempts and no additional radiation exposure to the patient. These studies suggest that preoperative registration coupled with direct navigation using procedure-specific graphical rendering, is potentially a highly accurate and effective means of performing image-guided interventions.

Cellular trafficking of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) play critical roles throughout the body. Precise regulation of the cellular location and availability of nAChRs on neurons and target cells is critical to their proper function. Dynamic, post-translational regulation of nAChRs, particularly control of their movements among the different compartments of cells, is an important aspect of that regulation. A combination of new information and new techniques has the study of nAChR trafficking poised for new breakthroughs.

Sustained cannabinoid agonist treatment augments CGRP release in a PKA-dependent manner.

Studies have shown that sustained cannabinoid treatment increases the sensitivity to painful heat stimuli (thermal hyperalgesia) and innocuous mechanical stimuli (tactile allodynia). It has been suggested that augmented release of pain neurotransmitters (such as calcitonin gene-related peptide, CGRP) might be responsible for this abnormal pain sensitization. We hypothesize that intracellular adaptations upon sustained cannabinoid treatment causes augmented release of CGRP from primary nociceptors leading to increased pain sensitivity. We show that sustained (24 h) cannabinoid agonist [(+)WIN 55,212-2] treatment of 7-day-old neonatal rat dorsal root ganglion neurons significantly augments basal CGRP release from these cells in a protein kinase A-dependent manner. Our results indicate that these intracellular compensatory adaptations may play a crucial trigger role in further neuronal system adaptations for modulation of pain.

Sustained morphine treatment augments basal CGRP release from cultured primary sensory neurons in a Raf-1 dependent manner.

Recent studies suggest that sustained morphine-mediated paradoxical pain may play an important role in the development of analgesic tolerance. The intracellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release are not fully clarified. Cyclic AMP (cAMP)-dependent protein kinase (PKA) plays an important role in the modulation of presynaptic neurotransmitter release. Moreover, we have shown earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). Therefore, in the present study we examined the role of Raf-1 in sustained morphine-mediated regulation of cAMP formation and basal CGRP release in vitro, in cultured neonatal rat dorsal root ganglion (DRG) neurons. We found that sustained morphine treatment significantly augments intracellular cAMP production as well as basal CGRP release from cultured neonatal rat DRG neurons. The selective PKA inhibitor, H-89, attenuates the sustained morphine-mediated augmentation of basal CGRP release, indicating that the cAMP/PKA pathway plays an important role in regulation of CGRP release from sensory neurons. Since our present data also demonstrated that selective Raf-1 inhibitor, GW 5074, attenuated both the cAMP overshoot and the augmentation of CGRP release mediated by sustained morphine in neonatal rat DRG neurons, we suggest that Raf-1-mediated sensitization of the intracellular cAMP formation may play an important role in sustained morphine-mediated augmentation of spinal pain neurotransmitter release.

Differential binding and activation of caspase-3 in cultured hippocampal neurons by assembly forms of A beta 1-42.

Amyloid-beta (A beta) peptides, the primary constituents of amyloid plaques in the brain in Alzheimer's disease (AD), may cause AD, but how they do so is not clear. A beta peptides spontaneously aggregate, or self-assemble, to generate several distinct macromolecular and morphological forms that can differ significantly in their effects on cells. We have compared different assembly forms of A beta(1-42) (A beta 42) for their ability to trigger apoptosis in cultured hippocampal neurons at a submicromolar concentration and for their binding to such neurons. Fibrillar A beta 42 caused both morphological changes indicative of apoptosis and specific activation of caspase-3, a characteristic marker of neurodegeneration in AD, in hippocampal neurons, whereas other preparations tested did not do so under the same conditions. More aggregated forms of A beta 42, including both fibrils and a mixture of assembly forms termed A beta-derived diffusible ligands (ADDLs), bound to neurons much more extensively and at lower concentrations than preparations that contained smaller forms. Fibrillar A beta 42, in particular, bound to neurons at concentrations as low as 1 nM. Colocalization studies showed that fibrillar A beta 42 bound almost exclusively at nonsynaptic sites. These results show differences between assembly forms of A beta 42 in the ability to trigger apoptotic signaling in CNS neurons, and they directly demonstrate differences between assembly forms in the binding to CNS neurons, a possible first step in the pathogenesis of AD. These results suggest that fibrillar A beta 42 contributes to the pathogenesis of AD.

Iptakalim modulates ATP-sensitive K(+) channels in dopamine neurons from rat substantia nigra pars compacta.

Iptakalim, a novel cardiovascular ATP-sensitive K(+) (K(ATP)) channel opener, exerts neuroprotective effects on dopaminergic (DA) neurons against metabolic stress-induced neurotoxicity, but the mechanisms are largely unknown. Here, we examined the effects of iptakalim on functional K(ATP) channels in the plasma membrane (pm) and mitochondrial membrane using patch-clamp and fluorescence-imaging techniques. In identified DA neurons acutely dissociated from rat substantia nigra pars compacta (SNc), both the mitochondrial metabolic inhibitor rotenone and the sulfonylurea receptor subtype (SUR) 1-selective K(ATP) channel opener (KCO) diazoxide induced neuronal hyperpolarization and abolished action potential firing, but the SUR2B-selective KCO cromakalim exerted little effect, suggesting that functional K(ATP) channels in rat SNc DA neurons are mainly composed of SUR1. Immunocytochemical staining showed a SUR1-rather than a SUR2B-positive reaction in most dissociated DA neurons. At concentrations between 3 and 300 microM, iptakalim failed to hyperpolarize DA neurons; however, 300 microM iptakalim increased neuronal firing. In addition, iptakalim restored DA neuronal firing during rotenone-induced hyperpolarization and suppressed rotenone-induced outward current, suggesting that high concentrations of iptakalim close neuronal K(ATP) channels. Furthermore, in human embryonic kidney 293 cells, iptakalim (300-500 microM) closed diazoxide-induced Kir6.2/SUR1 K(ATP) channels, which were heterologously expressed. In rhodamine-123-preloaded DA neurons, iptakalim neither depolarized mitochondrial membrane nor prevented rotenone-induced mitochondrial depolarization. These data indicate that iptakalim is not a K(ATP) channel opener in rat SNc DA neurons; instead, iptakalim is a pm-K(ATP) channel closer at high concentrations. These effects of iptakalim stimulate further pharmacological investigation and the development of possible therapeutic applications.

Roles of nicotinic acetylcholine receptor beta subunits in function of human alpha4-containing nicotinic receptors.

Naturally expressed nicotinic acetylcholine receptors (nAChR) containing alpha4 subunits (alpha4*-nAChR) in combination with beta2 subunits (alpha4beta2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. beta4 subunits are also richly expressed and colocalize with alpha4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, alpha4beta4-nAChR also may exist and play important functional roles. In this study, properties were determined of human alpha4beta2- and alpha4beta4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human alpha4beta4-nAChR have approximately 4-fold higher amplitude than those mediated via human alpha4beta2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at alpha4beta4-nAChR than at alpha4beta2-nAChR. Cytisine and lobeline serve as full agonists at alpha4beta4-nAChR but are only partial agonists at alpha4beta2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional alpha4beta2- and alpha4beta4-nAChR. Whole-cell current responses show stronger inward rectification for alpha4beta2-nAChR than for alpha4beta4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR beta2 or beta4 subunits can combine with alpha4 subunits to generate two forms of alpha4*-nAChR with distinctive physiological and pharmacological features. Diversity in alpha4*-nAChR is of potential relevance to nervous system function, disease, and nicotine dependence.

Electrophysiological properties of human hypothalamic hamartomas.

The hypothalamic hamartoma (HH) is a rare developmental malformation often characterized by gelastic seizures, which are usually refractory to medical therapy. The mechanisms of epileptogenesis operative in this subcortical lesion are unknown. In this study, we used standard patch-clamp electrophysiological techniques combined with histochemical approaches to study individual cells from human HH tissue immediately after surgical resection. More than 90% of dissociated HH cells were small (6-9 microm soma) and exhibited immunoreactivity to the neuronal marker NeuN, and to glutamic acid decarboxylase, but not to glial fibrillary acidic protein. Under current-clamp, whole-cell recordings in single dissociated cells or in intact HH slices demonstrated typical neuronal responses to depolarizing and hyperpolarizing current injection. In some cases, HH cells exhibited a "sag-like" membrane potential change during membrane hyperpolarization. Interestingly, most HH cells exhibited robust, spontaneous "pacemaker-like" action potential firing. Under voltage-clamp, dissociated HH cells exhibited functional tetrodotoxin (TTX)-sensitive Na(+) and tetraethylammonium-sensitive K(+) currents. Both GABA and glutamate evoked whole-cell currents, with GABA exhibiting a peak current amplitude 10-fold greater than glutamate. These findings suggest that human HH tissues, associated with gelastic seizures, contained predominantly small GABAergic inhibitory neurons that exhibited intrinsic "pacemaker-like" behavior.

Role of Nova-1 in regulating alpha2N, a novel glycine receptor splice variant, in developing spinal cord neurons.

Inhibitory glycine receptor (GlyR) subunits undergo developmental regulation, but the molecular mechanisms of GlyR regulation in developing neurons are little understood. Using RT-PCR, we investigated the regulation of GlyR alpha-subunit splice forms during the development of the spinal cord of the rat. Experiments to compare the amounts of mRNA for two known splice variants of the GlyR alpha2 subunit, alpha2A and alpha2B, in the developing rat spinal cord revealed the presence of an additional, novel variant that lacked any exon 3, herein named "alpha2N." Examination of the RNA from spinal cords of different-aged rats showed a dramatic down-regulation of alpha2N during prenatal development: alpha2N mRNA formed a significant portion of the alpha2 subunit pool at E14, but its relative level was reduced by 85% by birth and was undetectable in adults. Two proteins previously implicated in regulating the splicing of GlyR alpha2 pre-mRNA, the neurooncological ventral antigen-1 (Nova-1) and the brain isoform of the polypyrimidine tract binding protein (brPTB), underwent small changes over the same period that did not correlate directly with the changes in the level of alpha2N, calling into question their involvement in the developmental regulation of alpha2N. However, treatment of spinal cord neurons in culture with antisense oligonucleotides designed selectively to knock down one of three Nova-1 variants significantly altered the relative level of GlyR alpha2N, showing that Nova-1 isoforms can regulate GlyR alpha2 pre-mRNA splicing in developing neurons. These results provide evidence for a novel splice variant of the GlyR alpha2 subunit that undergoes dramatic developmental regulation, reveal the expression profiles of Nova-1 and brPTB in the developing spinal cord, and suggest that Nova-1 plays a role in regulating GlyR alpha2N in developing neurons.

Expression of alpha(2)-adrenergic receptor subtypes in prenatal rat spinal cord.

The results of molecular cloning have revealed three subtypes of the alpha(2)-adrenergic receptors (alpha(2) AR) that have been defined alpha(2)C10 (alpha(2A)), alpha(2)C2 (alpha(2B)) and alpha(2)C4 (alpha(2C)). The differential expression of alpha(2) AR subtypes is affected by developmental factors in rat submandibular gland, lung and brain. In the spinal cord of postnatal and adult rats, alpha(2A) and alpha(2C) AR subtypes are expressed and appear to mediate pain perception. However, the relative expression of alpha(2) AR subtypes in the prenatal spinal cord is unknown. In the present study subtype-specific antibodies and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the expression and localization of the alpha(2) AR subtypes in sections of embryonic day 14 rat spinal cords and primary cultures of cells isolated from these cords. Spinal cords were removed from day 14 embryos, and were sectioned or used for the preparation of cell cultures. After 9 days in culture, neurons were examined by immunofluorescence microscopy or used for preparation of total RNA. In both intact spinal cords and isolated cells, positive immunoreactivity was detected with antibodies against alpha(2A) and alpha(2B) subtypes, but not with antibodies against the alpha(2C) subtype. Using a dual-labeling approach, anti-alpha(2A) and anti-alpha(2B) immunoreactivity was present on the same population of neurons. RT-PCR results were consistent with immunofluorescence studies, and showed that mRNA encoding the alpha(2A) and alpha(2B) subtypes was present in total RNA prepared from primary cultures of rat spinal cord neurons. In contrast to spinal cords of postnatal or adult rats that express alpha(2A) and alpha(2C) AR subtypes on different neurons, prenatal spinal cords contain alpha(2A) and alpha(2B) AR subtypes, and these two subtypes appear to be co-expressed in the same cells.