Outcomes from an elective medical student Research Scholarly Concentration program.

To examine how to increase research career outcomes among medical graduates, we analyzed the impact of the Research Scholarly Concentration at The George Washington University School of Medicine and Health Sciences. Residency placement, subsequent scholarship, and career outcomes were compared among 670 graduates who participated in the elective Clinical and Translational Research Scholarly Concentration or no Concentration between 2009 and 2018. We conducted a retrospective study of residency match (highly selective vs less selective), job type (academic vs non-academic), and postmedical school publications (any vs none). We compared the outcomes between Research Scholarly Concentration graduates and those with no Concentration, matched by graduation year (n=335). For Research Scholarly Concentration graduates, we examined the association between research outcomes and duration of research experience before medical school (n=232). Research Scholarly Concentration graduates were more likely to place in a highly selective residency (40.2% vs 21.6%, p<0.0001), 68% more likely to publish after medical school (OR=1.68, 95% CI 1.10 to 2.58), and almost four times as likely to have taken an academic health center job (OR=3.82, 95% CI 1.72 to 8.46) than graduates with no Concentration. Surprisingly, the length of research experience before medical school was not associated with these outcomes among Research Scholarly Concentration graduates. This suggests that a medical school Research Scholarly Concentration is effective in training physician researchers and should be available to both novices and research-experienced matriculants. These data suggest how other medical schools might plan Scholarly Concentration programs to improve research outcomes among medical graduates.

Clinician-Investigator Training and the Need to Pilot New Approaches to Recruiting and Retaining This Workforce.

Clinician-investigators, also called physician-scientists, offer critical knowledge and perspectives that benefit research on basic science mechanisms, improved diagnostic and therapeutic approaches, population and outcomes medicine, health policy, and health services, yet few clinically trained health professionals pursue a research career. Sustaining this workforce requires attention to the unique challenges faced by investigators who must achieve clinical and research competence during training and their careers. These challenges include the duration of required clinical training, limited or discontinuous research opportunities, high levels of educational debt, balancing the dual obligations and rewards of clinical care and research, competition for research funding, and the need for leadership development after training. Women and individuals from underrepresented racial and ethnic groups comprise a small percentage of this workforce.The authors summarize the recent literature on training for clinician-investigators, emphasizing approaches with encouraging outcomes that warrant broader implementation. Using this overview as background, they convened three workshops at the National Institutes of Health in 2016 to identify and refine key priorities for potential new pilot programs to recruit and retain the clinician-investigator workforce. From these workshops emerged three priorities for future pilot programs: (1) support for research in residency, (2) new research on-ramps for health professionals at multiple career stages, and (3) national networks to diversify and sustain clinician-investigator faculty. Implementation of any pilot program will require coordinated commitment from academic health centers, medical licensing/certification boards, professional societies, and clinician-investigators themselves, in addition to support from the National Institutes of Health.

Delayed activin A administration attenuates tissue death after transient focal cerebral ischemia and is associated with decreased stress-responsive kinase activation.

Focal cerebral ischemia and reperfusion initiates complex cellular and molecular interactions that lead to either cell repair or destruction. In earlier work, we found that activin A is an early gene response to cerebral ischemia and supports cortical neuron survival in vitro. In this study, the ability of exogenous activin A to attenuate injury from transient middle cerebral artery occlusion was tested in adult mice. Intracerebroventricular administration of activin A prior to middle cerebral artery occlusion reduced infarct volume apparent 1 day after experimental stroke. A single activin A administration at 6 h following ischemia/reperfusion reduced lesion volumes at 1 and 3 days and led to improved neurobehavior. Moreover, activin A treatment spared neurons within the ischemic hemisphere and led to a concomitant reduction in microglial activation. Activation of the stress-responsive kinases p38 and c-jun N-terminal kinase implicated in neuronal apoptosis after stroke was reduced following activin A treatment. Together these findings suggest that activin A promotes tissue survival after focal cerebral ischemia/reperfusion with an extended therapeutic window.

Activin acutely sensitizes dorsal root ganglion neurons and induces hyperalgesia via PKC-mediated potentiation of transient receptor potential vanilloid I.

Pain hypersensitivity is a cardinal sign of tissue damage, but how molecules from peripheral tissues affect sensory neuron physiology is incompletely understood. Previous studies have shown that activin A increases after peripheral injury and is sufficient to induce acute nociceptive behavior and increase pain peptides in sensory ganglia. This study was designed to test the possibility that the enhanced nociceptive responsiveness associated with activin involved sensitization of transient receptor potential vanilloid I (TRPV1) in primary sensory neurons. Activin receptors were found widely distributed among adult sensory neurons, including those that also express the capsaicin receptor. Whole-cell patch-clamp recording from sensory neurons showed that activin acutely sensitized capsaicin responses and depended on activin receptor kinase activity. Pharmacological studies revealed that the activin sensitization of capsaicin responses required PKCepsilon signaling, but not PI3K (phosphoinositide 3-kinase), ERK (extracellular signal-regulated protein kinase), PKA, PKCalpha/beta, or Src. Furthermore, activin administration caused acute thermal hyperalgesia in wild-type mice, but not in TRPV1-null mice. These data suggest that activin signals through its own receptor, involves PKCepsilon signaling to sensitize the TRPV1 channel, and contributes to acute thermal hyperalgesia.

Bone morphogenetic proteins promote gliosis in demyelinating spinal cord lesions.

OBJECTIVE: To determine the role of bone morphogenetic proteins (BMPs) in stimulating glial scar formation in demyelinating lesions of the adult spinal cord. METHODS: The dorsal columns of adult rats were injected with lysolecithin to induce a local demyelinating lesion. Levels of BMP4 and BMP7 proteins were assayed and compared with glial fibrillary acidic protein expression in the injury area. BMP-responsive cells were identified by expression of phosphorylated Smad1/5/8. Cultures of mature spinal cord astrocytes were treated with BMP4, and levels of chondroitin sulphate proteoglycans (CSPGs) were measured. The effect of BMP4 on CSPG gene regulation was determined by real-time polymerase chain reaction for CSPG core proteins. RESULTS: BMP4 and BMP7 increase rapidly at the site of demyelination, and astrocytes surrounding the lesion increase expression of phosphorylated Smad1/5/8. Cultured mature astrocytes respond directly to BMPs with Smad1 translocation to the nucleus, increased phosphorylated Smad1/5/8, and increases in glial fibrillary acidic protein and CSPG expression. BMP treatment also increased CSPG messenger RNA for CSPG core proteins, including aggrecan and neurocan. Increases in CSPG expression in astrocytes by BMPs were blocked by the inhibitor noggin. Injections of BMP4 or BMP7 into the dorsal columns in the absence of demyelination led to increases in CSPG expression. INTERPRETATION: Local increases in BMPs at the site of a demyelinating lesion causes upregulation of gliosis, glial scar formation, and heightened expression of CSPGs such as neurocan and aggrecan that may inhibit remyelination.

Activin is a neuronal survival factor that is rapidly increased after transient cerebral ischemia and hypoxia in mice.

One approach for developing targeted stroke therapies is to identify the neuronal protective and destructive signaling pathways and gene expression that follow ischemic insult. In some neural injury models, the transforming growth factor-beta family member activin can provide neuroprotective effects in vivo and promote neuronal survival. This study tests if activin supports cortical neurons after ischemic challenge in vitro and if signals after cerebral ischemia involve activin in vivo. In a defined cell culture model that uses hydrogen peroxide (H(2)O(2))-free radical stress, activin addition maintained neuronal survival. H(2)O(2) treatment increased activin mRNA twofold in surviving cortical neurons, and inhibition of activin with neutralizing antibodies caused neuronal death. These data identify activin gene changes as a rapid response to oxidative stress, and indicate that endogenous activin acts as a protective factor for cortical neurons in vitro. Similarly, after transient focal cerebral ischemia in adult mice, activin mRNA increased at 1 and 4 h ipsilateral to the infarct but returned to control values at 24 h after reperfusion. Intracellular activated smad signals were detected in neurons adjacent to the infarct. Activin was also increased after 2 h of 11% hypoxia. Activin mRNA increased at 1 h but not 4 or 24 h after hypoxia, similar to the time course of erythropoietin and vascular endothelial growth factor induction. These findings identify activin as an early-regulated gene response to transient ischemia and hypoxia, and its function in cortical neuron survival during oxidative challenge provides a basis to test activin as a potential therapeutic in stroke injury.

The role of activin in neuropeptide induction and pain sensation.

Signals from target tissues play critical roles in the functional differentiation of neuronal cells, and in their subsequent adaptations to peripheral changes in the adult. Sensory neurons in the dorsal root ganglia (DRG) provide an excellent model system for the study of signals that regulate the development of neuronal diversity. DRG have been well characterized and contain both neurons that convey information from muscles about limb position, as well as other neurons that provide sensations from skin about pain information. Sensory neurons involved in pain sensation can be distinguished physiologically and antigenically, and one hallmark characteristic is that these neurons contain neuropeptides important for their functions. The transforming growth factor (TGF) beta family member activin A has recently been implicated in neural development and response to injury. During sensory neuron development, peripheral target tissues containing activin or activin itself can regulate pain neuropeptide expression. Long after development has ceased, skin target tissues retain the capacity to signal neurons about changes or injury, to functionally refine synapses. This review focuses on the role of activin as a target-derived differentiative factor in neural development that has additional roles in response to cutaneous injuries in the adult.

Reduced expression of endothelin B receptors and mechanical hyperalgesia in experimental chronic diabetes.

Diabetic neuropathy is one of the major complications of diabetes mellitus. Small nerve fibers degenerate early in the disease, leading to symptoms ranging from hyperalgesia to loss of pain and temperature sensation. However, the cellular and molecular mechanisms responsible for abnormal pain perception in diabetes have not been identified. Both type-A and type-B endothelin receptors (ETAR and ETBR, respectively) are present in sensory nerves and appear to regulate neuropathic and inflammatory pain. In this study, we compared the expression of endothelin receptors and nociceptive responses in normal and experimentally diabetic rats. Diabetic animals exhibited both an increase in the withdrawal responses to high threshold stimuli (mechanical hyperalgesia) and to light touch stimuli (tactile allodynia). Immunohistochemical and Western blot analysis revealed that diabetic rats have significantly reduced expression of ETBR in sciatic nerves, while no changes were observed in dorsal root ganglia (DRG). In contrast, the expression of ETAR in either sciatic nerves or DRG of diabetic rats was not altered. Importantly, ETBR-deficient transgenic rats showed alterations in pain perception similar to those observed in diabetic rats. These results suggest that changes in the expression of ETBR in peripheral nerve may contribute to the development of mechanical hyperalgesia and tactile allodynia in chronic diabetes.

Rodent sensory neuron culture and analysis.

Sensory neurons have proven very useful for analysis of neuronal differentiation in vivo and in vitro. Their utility for in vitro work is based on the fact that sensory neurons are relatively easy to isolate in large numbers and are amenable to manipulations in culture. Lumbar ganglia are usually used because their location in the caudal nervous system means they are the least differentiated at any developmental stage, allowing the analysis of relatively undifferentiated cells. Rodent sensory ganglia from embryonic to adult stages can be dissected effectively and maintained in serum-free medium or in coculture with other cells or factors. This unit describes generation of embryonic rat lumbar dorsal root ganglia (DRG) cultures, which form an important model system for investigating the cellular and molecular mechanisms that regulate neuronal differentiation. Adult DRG can also be successfully cultured, with a few modifications of the general protocol.

Activin induces tactile allodynia and increases calcitonin gene-related peptide after peripheral inflammation.

Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide important in inflammatory pain that conveys pain information centrally and dilates blood vessels peripherally. Previous studies indicate that activin A increases CGRP-immunoreactive (IR) sensory neurons in vitro, and following wound, activin A protein increases in the skin and more neurons have detectable CGRP expression in the innervating dorsal root ganglion (DRG). These data suggest some adult sensory neurons respond to activin A or other target-derived factors with increased neuropeptide expression. This study was undertaken to test whether activin contributes to inflammatory pain and increased CGRP and to learn which neurons retained plasticity. After adjuvant-induced inflammation, activin mRNA, but not NGF or glial cell line-derived neurotrophic factor, increased in the skin. To examine which DRG neurons increased CGRP immunoreactivity, retrograde tracer-labeled cutaneous neurons were characterized after inflammation. The proportion and size of tracer-labeled DRG neurons with detectable CGRP increased after inflammation. One-third of CGRP-IR neurons that appear after inflammation also had isolectin B4 binding, suggesting that some mechanoreceptors became CGRP-IR. In contrast, the increased proportion of CGRP-IR neurons did not appear to come from RT97-IR neurons. To learn whether central projections were altered after inflammation, CGRP immunoreactivity in the protein kinase Cgamma-IR lamina IIi was quantified and found to increase. Injection of activin A protein alone caused robust tactile allodynia and increased CGRP in the DRG. Together, these data support the hypothesis that inflammation and skin changes involving activin A cause some sensory neurons to increase CGRP expression and pain responses.

Wounds increase activin in skin and a vasoactive neuropeptide in sensory ganglia.

Successful healing of skin wounds requires sensory innervation and the release of vasoactive neuropeptides that dilate blood vessels and deliver serum proteins to the wound, and that cause pain that protects from further injury. Activin has been proposed as a target-derived regulator of sensory neuropeptides during development, but its role in the mature nervous system is unknown. While adult skin contains a low level of activin, protein levels in skin adjacent to a wound increase rapidly after an excision. Neurons containing the neuropeptide calcitonin gene-related peptide (CGRP) increased in sensory ganglia that projected to the wounded skin, but not in ganglia that projected to unwounded skin, suggesting that neurons respond to a local skin signal. Indeed, many adult sensory neurons respond with increased CGRP expression to the application of activin in vitro and utilize a smad-mediated signal transduction pathway in this response. A second skin-derived factor nerve growth factor (NGF) also increased in wounded skin and increased CGRP in cultured adult dorsal root ganglia (DRG) neurons but with lower efficacy. Together, these data support the hypothesis that activin made by skin cells regulates changes in sensory neuropeptides following skin injury, thereby promoting vasodilation and wound healing.

Emerging roles for bone morphogenetic proteins in central nervous system glial biology.

Bone morphogenetic proteins, members of the TGFbeta superfamily have been implicated in a variety of roles in the developing and mature nervous system. These divergent functions are a reflection of the closely defined spatial and temporal expression of BMPs in the CNS, and the potential interactions of the BMP signaling pathway with the STAT and MAP kinase pathways. In this review we discuss the roles of BMPs in early patterning of the CNS, determination of neural cell fate, and regulation of oligodendrocyte maturation during CNS development. Additional functions for members of the TGFbeta superfamily in CNS injury responses are emerging suggesting these molecules represent useful targets for manipulating neural responses to CNS insults.

Patterning of spinal cord oligodendrocyte development by dorsally derived BMP4.

Oligodendrocyte precursors (OPCs) initially arise in the motor neuron domain of the ventral ventricular zone of the developing spinal cord. After dispersal throughout gray and white matter, OPCs differentiate in a characteristic ventral to dorsal sequence. The spatial localization of OPC induction is in part a result of both positive local sonic hedgehog signaling and dorsally derived inhibitory cues. One component of dorsal inhibitory signals seems to be members of the transforming growth factor beta (TGFbeta) superfamily such as the bone morphogenetic proteins (BMPs). We show that during the initial appearance and subsequent maturation of OPCs, BMP4 was expressed specifically in the dorsal midline and its expression was correlated spatially and temporally with phospho-Smad 1+, BMP4-responsive cells. Implantation of sonic hedgehog (Shh)-coated beads adjacent to dorsal spinal cord in Xenopus embryos induced ectopic dorsal OPCs whereas BMP4-coated beads inhibited OPC appearance. More importantly, blocking endogenous dorsal BMP4 with anti-BMP4-coated beads locally induced ectopic OPCs. Similar results were obtained using soluble ligands on slice preparations of rodent spinal cord in vitro. In dissociated cell cultures of embryonic rat spinal cord, Shh and BMP4 had antagonistic effects on OPC development and the sensitivity of oligodendrocyte lineage cells to BMP4 increased with maturation. These data suggest that BMP4 contributes to the pattern of spinal cord oligodendrogenesis by regulating both induction and maturation of spinal cord OPCs.

Signaling by bone morphogenetic proteins and Smad1 modulates the postnatal differentiation of cerebellar cells.

Previous studies have demonstrated that bone morphogenetic proteins (BMPs) activate the Smad1 signaling pathway to regulate cell determination and differentiation in the embryonic nervous system. Studies examining gene and protein expression in the rat cerebellum suggest that this pathway also regulates postnatal differentiation. Using microarrays, we found that Smad1 mRNA expression in the cerebellum increases transiently at postnatal day 6 (P6). Immunohistochemistry and Western blots showed that Smad1 and BMP4 proteins are present in the cerebellum, and that their expression also changes postnatally. The proteins are detectable at P4-P6, a stage at which most cerebellar cells reside in the external germinal layer (EGL), where they extensively differentiate. The levels become maximal at P8-P10, when neurons begin to migrate from the EGL into their mature positions in the internal granule layer. In cerebellar cultures prepared at P6 or P10, BMP4 activates Smad1 signaling to modulate cell differentiation. Brief BMP4 application caused Smad1 translocation from the neuronal cytoplasm into the nucleus, where it is known to regulate transcription in association with Smad4. Longer BMP4 treatment promoted the differentiation of both neuronal and non-neuronal cells. By 3 d, neuronal processes appeared more fasciculated, and the level of synaptotagmin, a protein found in synaptic vesicles, increased. In addition, many astroglial cells became more branched and stellate in morphology. The BMP-induced changes were reduced by treatment with antisense oligonucleotides to Smad1 or Smad4. These findings in vivo and in culture suggest that BMP4 and Smad1 signaling participate in regulating postnatal cerebellar differentiation.

Activin and bone morphogenetic proteins are present in perinatal sensory neuron target tissues that induce neuropeptides.

Previous studies have shown that sensory target tissues induce neuropeptides in naïve sensory neurons, and that activin and bone morphogenetic proteins (BMPs) are capable of inducing neuropeptides associated with nociception in embryonic sensory neurons in vitro. The goal of the present study was to learn if these ligands were available in native sensory neuron target tissues at correct developmental periods to play this inductive role in vivo. Sensory neurons initially contact their peripheral target tissues and begin to express neuropeptides during late embryogenesis, and we demonstrate that activin and BMPs are present in the embryo and neonate to regulate sensory neuron differentiation. Native embryonic and neonatal target tissues were analyzed by immunoblot and immunohistochemical studies using ligand-specific antibodies. Although activin was easily solubilized, BMPs were detected only after high salt extraction, suggesting that BMPs were bound to extracellular moieties and were capable of acting only locally in native tissues. One inhibitor, noggin, was present in both embryonic skin and muscle. In combination, these data suggest that neuronal differentiation is unlikely to be regulated by simple expression of ligand, but that the functional availability of ligand is a critical component confering biological activity.