Socioeconomic status is associated with greater hazard of post-discharge mortality than race, gender, and ballistic injury mechanism in a young, healthy, orthopedic trauma population.

OBJECTIVES: To explore the utility of legacy demographic factors and ballistic injury mechanism relative to popular markers of socioeconomic status as prognostic indicators of 10-year mortality following hospital discharge in a young, healthy patient population with isolated orthopedic trauma injuries. METHODS: A retrospective cohort study was performed to evaluate patients treated at an urban Level I trauma center from January 1, 2003, through December 31, 2016. Current Procedure Terminology (CPT) codes were used to identify upper and lower extremity fracture patients undergoing operative fixation. Exclusion criteria were selected to yield a patient population of isolated extremity trauma in young, otherwise healthy individuals between the ages of 18 and 65 years. Variables collected included injury mechanism, age, race, gender, behavior risk factors, Area Deprivation Index (ADI), and insurance status. The primary outcome was post-discharge mortality, occurring at any point during the study period. RESULTS: We identified 2539 patients with operatively treated isolated extremity fractures. The lowest two quartiles of socioeconomic status (SES) were associated with higher hazard of mortality than the highest SES quartile in multivariable analysis (Quartile 3 HR: 2.2, 95% CI: 1.2-4.1, p = 0.01; Quartile 4 HR: 2.2, 95% CI: 1.1-4.3, p = 0.02). Not having private insurance was associated with higher mortality hazard in multivariable analysis (HR 2.0, 95% CI: 1.3-3.2, p = 0.002). The presence of any behavioral risk factor was associated with higher mortality hazard in univariable analysis (HR: 1.8, p < 0.05), but this difference did not reach statistical significance in multivariable analysis (HR: 1.4, 95%: 0.8-2.3, p = 0.20). Injury mechanism (ballistic versus blunt), gender, and race were not associated with increased hazard of mortality (p > 0.20). CONCLUSION: Low SES is associated with a greater hazard of long-term mortality than ballistic injury mechanism, race, gender, and medically diagnosable behavioral risk factors in a young, healthy orthopedic trauma population with isolated extremity injury.

Preoperative Factors Associated With Worse PROMIS Pain Interference 2 Years After Hand and Wrist Surgery.

BACKGROUND: The purpose of this study was to identify preoperative factors associated with worse postoperative Patient-Reported Outcomes Measurement Information System (PROMIS) pain interference (PI) scores 2 years after hand and wrist surgery. We hypothesized that older age, more comorbidities, increased substance use, and lower socioeconomic status would correlate with worse 2-year PROMIS PI scores. METHODS: This study was a retrospective review of prospectively acquired data on 253 patients. Surveys were administered within 1 week of surgery and 2 years postoperatively. Bivariate and multivariable analyses were conducted to identify significant predictors of worse 2-year PROMIS PI scores and change in PROMIS PI scores. RESULTS: Older age, higher body mass index, more comorbidities, lower preoperative expectations, more prior surgeries, unemployment, smoking, higher American Society of Anesthesiologists (ASA) score, and multiple other socio-demographic factors were correlated with worse 2-year PROMIS PI scores (P ≤ .018). Similar factors were also correlated with less improvement in 2-year PROMIS PI scores (P ≤ .048). Worse scores on all preoperative patient-reported outcome measures correlated with worse 2-year PROMIS PI scores (P ≤ .007). Multivariable analysis identified smoking history, less frequent alcohol consumption, worse preoperative PROMIS social satisfaction and Numeric Pain Scale whole body scores, and higher ASA scores as independent predictors of worse 2-year PROMIS PI. The same factors in addition to better baseline PROMIS PI were predictive of less improvement in 2-year PROMIS PI. CONCLUSION: Numerous preoperative factors were predictive of worse postoperative 2-year PROMIS PI and less improvement in 2-year PROMIS PI for patients undergoing hand and wrist surgery.

Small-Fragment Plate Fixation of Humeral Shaft Fractures.

Fixation of humeral shaft fractures is frequently performed with large-fragment (4.5 mm) plates to accommodate immediate weight bearing. Use of small-fragment (3.5 mm) plates as an alternative carries theoretical benefits. We examined nonunion rates and postoperative radial nerve palsy (RNP) rates in a retrospective cohort of patients undergoing open reduction and internal fixation of humeral shaft fractures with 3.5-mm or 4.5-mm plates. Two hundred thirty-six patients with 241 humeral shaft fractures were included. Small 3.5-mm plates were used in 83% of the patients, and large 4.5-mm plates were used in 17% of the patients. Fifty-three percent were made weight bearing as tolerated following surgical fixation. There was a 7% incidence of nonunion and a 10% incidence of RNP in the 3.5-mm plate group. There was a 7% incidence of nonunion and a 15% incidence of RNP in the 4.5-mm plate group. No statistically significant relationship was shown between nonunion or RNP and plate size (P=.74 and P=.39). No relationship was shown between nonunion and postoperative weight-bearing status (P=.45). Subgroup analysis according to plate size additionally showed no association of nonunion with postoperative weight bearing in both the 4.5-mm (P=.55) and the 3.5-mm (P=.25) cohorts. Small-fragment and large-fragment plating of humeral shaft fractures resulted in comparable union and RNP rates, regardless of postoperative weight-bearing status. Our findings suggest that 3.5-mm plate fixation of humeral shaft fractures is a safe alternative to 4.5-mm plate fixation. [Orthopedics. 2023;46(4):198-204.].

Iatrogenic thrombosis of the deep inferior epigastric artery during diagnostic angiography-a rare complicating factor during rectus free flap harvest.

We describe a 28-year-old man who sustained an open IIIB left ankle fracture dislocation with heel pad avulsion. The patient underwent formal angiography of the left lower extremity, followed by free tissue transfer of a rectus abdominis flap several days later. Intraoperatively, a thrombus was identified in the deep inferior epigastric artery above the femoral artery access site requiring thrombectomy. Histologic analysis estimated the thrombus age at 12 to 72 hours, raising concern that the thrombus was induced during angiogram instrumentation. Donor and recipient site-specific risks of arterial instrumentation (including invasive diagnostics) should be considered when planning free tissue transfer.

Association of Radial Head Fracture Treatment With Long-Term Function.

BACKGROUND: Radial head fractures are often associated with poor outcomes. Both open reduction and internal fixation (ORIF) and radial head arthroplasty (RHA) might be considered in operative cases. This study aimed to compare long-term patient-reported functional outcomes among patients with operatively treated radial head fractures. METHODS: A cross sectional study conducted at a Level I trauma center was used to identify patients with a radial head fracture who underwent ORIF or RHA between 2006 and 2018, and agreed to complete a survey in 2020. The primary outcome measure was the Quick Disabilities of the Arm, Shoulder, and Hand (QuickDASH) score. RESULTS: Seventy-six patients participated in the study. No significant differences in outcomes were observed between groups. QuickDASH scores were similar for both groups (ORIF: mean = 15.7, SD = 18.4; RHA: mean = 22.8, SD = 18.6; mean difference = 0.2 [-9.0 to 9.3], P = .97). Nineteen (37%) ORIF patients and 12 (48%) RHA patients reported a need for pain medication (adjusted odds ratio [OR] = 0.8 [0.3-2.4], P = .70). Thirteen (25%) ORIF patients and 6 (24%) RHA patients required additional surgery (adjusted OR = 1.7 [0.5-6.2], P = .39). A subgroup analysis of multi-fragmentary fractures revealed similar findings. CONCLUSION: Patient-reported outcomes, which included a subgroup analysis of multi-fragmentary fractures, were similar between ORIF and RHA groups at an average of 7.5 years from surgery. Reconstructing the radial head might not result in worse outcomes than RHA when both options are employed according to the best judgment of the operating surgeon.

Telemedicine and Socioeconomics in Orthopaedic Trauma Patients: A Quasi-Experimental Study During the COVID-19 Pandemic.

INTRODUCTION: Socioeconomic factors may introduce barriers to telemedicine care access. This study examines changes in clinic absenteeism for orthopaedic trauma patients after the introduction of a telemedicine postoperative follow-up option during the COVID-19 pandemic with attention to patient socioeconomic status (SES). METHODS: Patients (n = 1,060) undergoing surgical treatment of pelvic and extremity trauma were retrospectively assigned to preintervention and postintervention cohorts using a quasi-experimental design. The intervention is the April 2020 introduction of a telemedicine follow-up option for postoperative trauma care. The primary outcome was the missed visit rate (MVR) for postoperative appointments. We used Poisson regression models to estimate the relative change in MVR adjusting for patient age and sex. SES-based subgroup analysis was based on the Area Deprivation Index (ADI) according to home address. RESULTS: The pre-telemedicine group included 635 patients; the post-telemedicine group included 425 patients. The median MVR in the pre-telemedicine group was 28% (95% confidence interval [CI], 10% to 45%) and 24% (95% CI, 6% to 43%) in the post-telemedicine group. Low SES was associated with a 40% relative increase in MVR (95% CI, 17% to 67%, P < 0.001) compared with patients with high SES. Relative MVR changes between pre-telemedicine and post-telemedicine groups did not reach statistical significance in any socioeconomic strata (low ADI, -6%; 95% CI, -25% to 17%; P = 0.56; medium ADI, -18%; 95% CI, -35% to 2%; P = 0.07; high ADI, -12%; 95% CI, -28% to 7%; P = 0.20). CONCLUSIONS: Low SES was associated with a higher MVR both before and after the introduction of a telemedicine option. However, no evidence in this cohort demonstrated a change in absenteeism based on SES after the introduction of the telemedicine option. Clinicians should be reassured that there is no evidence that telemedicine introduces additional socioeconomic bias in postoperative orthopaedic trauma care. LEVEL OF EVIDENCE: III.

Analysis of Computed Tomography-Based Technique for Measuring Femoral Anteversion: Implications for Measuring Rotation After Femoral Intramedullary Nail Insertion.

OBJECTIVES: To assess the reliability of the current computed tomography (CT)-based technique for determining femoral anteversion and quantify the prevalence and magnitude of side-to-side differences. DESIGN: Cross-sectional cohort study. SETTING: Academic trauma center. PATIENTS: We reviewed CT scans from 120 patients with bilateral full-length axial cuts of both femurs. Two hundred forty femurs with no fractures or other identifying features in their femora were included. Ten unique data sets were created to measure anteversion of the left and right sides. MAIN OUTCOME MEASUREMENTS: Intraobserver and interobserver reliability were calculated using intraclass correlation coefficients (ICCs) and pooled absolute differences. The mean absolute difference between the sides was determined using a fixed-effects model. RESULTS: Interobserver reliability was high (ICC: 0.85, 95% confidence interval [CI]: 0.83-0.88). The pooled mean absolute magnitude of variation between reviewers was small at 1.6 degrees (95% CI: 1.4-1.8 degrees) per scan. The intraobserver reproducibility was high (ICC: 0.91, 95% CI: 0.88-0.93) with a mean error of 2.7 degrees (95% CI: 2.2-3.1 degrees) per repeat viewing of the same scan by the same person. The magnitude of side-to-side variation was 2.0 degrees (95% CI: 1.5-2.6 degrees). Twenty-one subjects (18%, 95% CI: 12%-25%) had a mean side-to-side calculated femoral anteversion difference of ≥10 degrees, whereas 6 (5%, 95% CI: 2-10) subjects had a calculated mean side-to-side difference of ≥15 degrees. CONCLUSIONS: CT based femoral anteversion measurement techniques demonstrate good precision. Only 1 in 20 patients had side-to-side differences of 15 degrees or more.

Management of vasopressor induced ischemia.

PURPOSE: We evaluated a cohort of patients who developed vasopressor-induced limb ischemia and the management options to prevent progression or minimize morbidity of digital necrosis. METHODS: We reviewed all current literature on pressor-induced limb ischemia and report options for the management of patients requiring vasopressors who developed limb ischemia. We then retrospectively reviewed presentation, treatment, and short-term outcomes for patients at our tertiary referral academic medical center that developed this complication. Finally, we recommend guidelines for the tiered management of these complex patients. RESULTS: Thirty-six patients were included. Twenty-six patients (72%) required resuscitation with more than one vasopressor. Vasopressors were initiated for septic-shock (52.7%), cardiogenic-shock (16.7%), hypovolemic-shock (13.9%), acute transplant rejection (13.9%), and neurogenic-shock (2.8%). According to the tiered management recommendations, patients were managed with phase 1 care (19%), phase 2 care (8.3%), phase 3 care (50%) or phase 4 care (5.6%). The patient expired in the acute setting in 13.9% of cases. CONCLUSION: Life-saving vasopressors risk digital ischemia and necrosis. Early recognition, reporting, and treatment of this complication are important in minimizing morbidity. Using a tiered approach helps organize the healthcare team's management of this iatrogenic complication while respecting the treatment paradigm of "life over limb," and may be safely performed with acceptable outcomes.

The Serially-Operated Essex-Lopresti Injury: Long-Term Outcomes in a Retrospective Cohort.

Objective The main aim of this article is to report 10-year outcomes after Essex-Lopresti injury (ELI). Study Design Retrospective case series. Two level I trauma centers and one associated community hospital from 2003 to 2016. Patients Sixteen patients who sustained an ELI and were treated at one of our three regional hospitals. Intervention Initially, 4 patients (25%) were treated nonoperatively by immobilization and 12 patients (75%) were treated operatively. Proximal surgery included radial head open reduction and internal fixation (ORIF), radial head arthroplasty, radial head excision and forearm ORIF, and wound debridement. Ten patients (63%) were acutely identified with longitudinal forearm instability. Of these, four patients had the distal radioulnar joint pinned. In the other six patients, the forearm was immobilized. Overall, 16 patients underwent a total of 32 revision surgeries. Main Outcome Measure Performance of Patient-Reported Outcomes Measurement Information System Upper Extremity (PROMIS UE) Physical score, Numeric Rating Scale (NRS) score for pain-severity, and NRS score for satisfaction of overall outcome. Results Follow-up for outcome evaluation was available for 10 patients, at a median of 10 (interquartile range [IQR]: 8.0-12) years after date of injury. The median PROMIS UE Physical score was 36 (IQR: 33-38). Median NRS score for pain-severity on average was 5 (IQR: 0-6). The median NRS score for satisfaction of overall outcome was 7 (IQR: 5-8). Conclusion Patients who sustain an ELI generally have substantial deficits of upper extremity function as measured by PROMIS UE. Early radial head arthroplasty may be beneficial, but further study in a larger cohort is needed. Outcomes of nonoperative treatment and operative treatment were similar and suggest that current surgical treatments are incomplete.

Nerve Diameter in the Hand: A Cadaveric Study.

BACKGROUND: Nerve injuries in the hand are common and often pose a challenge for the upper extremity surgeon. A range of reconstructive options exist for nerve repair, but proper matching of nerve diameter is important for many of these techniques. The purposes of this study were to (1) describe the nerve diameters of the hand and their relative differences and (2) investigate whether there is a relationship between nerve diameter and external hand dimensions or body mass index. METHODS: We utilized 18 freshly frozen cadaveric hands from adult donors aged 20-86 of both sexes for this study. Two independent observers measured each nerve diameter to the nearest 0.1 mm using a digital caliper. RESULTS: Using the flexor zones as boundaries, a total of 33 nerve measurements were performed for each cadaveric hand. Nerve diameter increased from the distal to the proximal flexor zones. The internal common digital nerves in flexor zone 3 were larger than the external digital nerves. The median nerve was found to be nearly 2 times larger than the ulnar nerve at 2 locations within the wrist. There was a positive correlation between body mass index, hand span, hand width, and nerve diameter at several measured locations. CONCLUSIONS: This study provides reference values for nerve diameters of the hand and wrist and describes their relative differences. It is important for surgeons to be aware of these differences and to consider this information as we advance our efforts to reconstruct the hand and develop technologies for nerve repair.

Assessing effects on dendritic arborization using novel Sholl analyses.

Determining the shape of cell-specific dendritic arbors is a tightly regulated process that occurs during development. When this regulation is aberrant, which occurs during disease or injury, alterations in dendritic shape result in changes to neural circuitry. There has been significant progress on characterizing extracellular and intrinsic factors that regulate dendrite number by our laboratory and others. Generally, changes to the dendritic arbor are assessed by Sholl analysis or simple dendrite counting. However, we have found that this general method often overlooks local changes to the arbor. Previously, we developed a program (titled Bonfire) to facilitate digitization of neurite morphology and subsequent Sholl analysis and to assess changes to root, intermediate, and terminal neurites. Here, we apply these different Sholl analyses, and a novel Sholl analysis, to uncover previously unknown changes to the dendritic arbor when we overexpress an important regulator of dendrite branching, cytosolic PSD-95 interactor (cypin), at two developmental time points. Our results suggest that standard Sholl analysis and simple dendrite counting are not sufficient for uncovering local changes to the dendritic arbor.

A topographically modified substrate-embedded MEA for directed myotube formation at electrode contact sites.

Myoblast fusion into functionally distinct myotubes, and their subsequent integration with the nervous system, is a poorly understood phenomenon with important applications in basic science research, skeletal muscle tissue engineering, and cell-based biosensor development. We have previously demonstrated the ability of microelectrode arrays (MEAs) to record the extracellular action potentials of myotubes, and we have shown that this information reveals the presence of multiple, electrophysiologically independent myotubes even in unstructured cultures where there is extensive physical contact between cells (Langhammer et al., Biotechnol Prog 27:891-895, 2011). In this paper, we explore the ability of microscale topographical trenches to guide the myoblast alignment and fusion processes and use our findings to create a substrate-embedded MEA containing topographical trenches that are able to direct myotube contractility to specific locations. By combining substrate-embedded MEA technology with topographical patterns, we have developed a lab-on-a-chip test bed for the non-invasive examination of myotubes.

Skeletal myotube integration with planar microelectrode arrays in vitro for spatially selective recording and stimulation: a comparison of neuronal and myotube extracellular action potentials.

Microelectrode array (MEA) technology holds tremendous potential in the fields of biodetection, lab-on-a-chip applications, and tissue engineering by facilitating noninvasive electrical interaction with cells in vitro. To date, significant efforts at integrating the cellular component with this detection technology have worked exclusively with neurons or cardiac myocytes. We investigate the feasibility of using MEAs to record from skeletal myotubes derived from primary myoblasts as a way of introducing a third electrogenic cell type and expanding the potential end applications for MEA-based biosensors. We find that the extracellular action potentials (EAPs) produced by spontaneously contractile myotubes have similar amplitudes to neuronal EAPs. It is possible to classify myotube EAPs by biological signal source using a shape-based spike sorting process similar to that used to analyze neural spike trains. Successful spike-sorting is indicated by a low within-unit variability of myotube EAPs. Additionally, myotube activity can cause simultaneous activation of multiple electrodes, in a similar fashion to the activation of electrodes by networks of neurons. The existence of multiple electrode activation patterns indicates the presence of several large, independent myotubes. The ability to identify these patterns suggests that MEAs may provide an electrophysiological basis for examining the process by which myotube independence is maintained despite rapid myoblast fusion during differentiation. Finally, it is possible to use the underlying electrodes to selectively stimulate individual myotubes without stimulating others nearby. Potential uses of skeletal myotubes grown on MEA substrates include lab-on-a-chip applications, tissue engineering, co-cultures with motor neurons, and neural interfaces.

Neuroprotective drug riluzole amplifies the heat shock factor 1 (HSF1)- and glutamate transporter 1 (GLT1)-dependent cytoprotective mechanisms for neuronal survival.

Heat shock factor 1 (HSF1) mediates the cellular response to stress to increase the production of heat shock protein (HSP) chaperones for proper protein folding, trafficking, and degradation; failure of this homeostatic mechanism likely contributes to neurodegeneration. We show that the neuroprotective drug riluzole increased the amount of HSF1 in NG108-15 neuroprogenitor cells by slowing the specific turnover of HSF1 and supporting a more robust and sustained activation of HSF1. Using Hsp70-luciferase as a functional readout of the activity of HSF1, we show that riluzole amplified the heat shock induction of the reporter gene with an optimal increase at 1 µM. Immunocytochemical staining and Western blot quantitation of HSP70 in NG108-15 neuroprogenitor cells and embryonic spinal cord neurons provided corroborative evidence that riluzole amplified the HSF1-dependent regulation of HSP70 expression. Parallel studies on the GLT1 glutamate transporter showed that riluzole increased GLT1-reporter and GLT1 protein expression and that the increase was enhanced by heat shock and coincident with the increased expression of HSP70 and HSP90. This result is consistent with the anti-glutamatergic profile of riluzole and the presence of multiple heat shock elements on the GLT1 gene promoter, suggesting that riluzole may modulate GLT1 expression through HSF1. The increased HSP chaperones and GLT1 transporter blunted glutamate-induced and N-methyl D-aspartate receptor-mediated excitotoxic death. In summary, we show that riluzole increased the amount and activity of HSF1 to boost the expression of HSPs and GLT1 for neuroprotection under stress.

Automated Sholl analysis of digitized neuronal morphology at multiple scales.

Neuronal morphology plays a significant role in determining how neurons function and communicate. Specifically, it affects the ability of neurons to receive inputs from other cells and contributes to the propagation of action potentials. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease, schizophrenia, and mental retardation. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction. Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations. The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

Automated Sholl analysis of digitized neuronal morphology at multiple scales: Whole cell Sholl analysis versus Sholl analysis of arbor subregions.

The morphology of dendrites and the axon determines how a neuron processes and transmits information. Neurite morphology is frequently analyzed by Sholl analysis or by counting the total number of neurites and branch tips. However, the time and resources required to perform such analysis by hand is prohibitive for the processing of large data sets and introduces problems with data auditing and reproducibility. Furthermore, analyses performed by hand or using course-grained morphometric data extraction tools can obscure subtle differences in data sets because they do not store the data in a form that facilitates the application of multiple analytical tools. To address these shortcomings, we have developed a program (titled "Bonfire") to facilitate digitization of neurite morphology and subsequent Sholl analysis. Our program builds upon other available open-source morphological analysis tools by performing Sholl analysis on subregions of the neuritic arbor, enabling the detection of local level changes in dendrite and axon branching behavior. To validate this new tool, we applied Bonfire analysis to images of hippocampal neurons treated with 25 ng/ml brain-derived neurotrophic factor (BDNF) and untreated control neurons. Consistent with prior findings, conventional Sholl analysis revealed that global exposure to BDNF increases the number of neuritic intersections proximal to the soma. Bonfire analysis additionally uncovers that BDNF treatment affects both root processes and terminal processes with no effect on intermediate neurites. Taken together, our data suggest that global exposure of hippocampal neurons to BDNF results in a reorganization of neuritic segments within their arbors, but not necessarily a change in their number or length. These findings were only made possible by the neurite-specific Sholl data returned by Bonfire analysis.

Regulation of dendrite arborization by substrate stiffness is mediated by glutamate receptors.

Brain injury or disease can initiate changes in local or global stiffness of brain tissue. While stiffness of the extracellular environment is known to affect the morphology and function of many cell types, little is known about how the dendrites of neurons respond to changes in brain stiffness. To assess how extracellular stiffness affects dendrite morphology, we took biomaterial and biomedical engineering approaches. We cultured mixed and pure hippocampal neurons on hydrogels composed of polyacrylamide (PA) of varying stiffnesses to mimic the effects of extracellular matrix stiffness on dendrite morphology. The majority of investigations of cortical and spinal cord neurons on soft hydrogels examined branching at early time points (days in vitro (DIV) 2-7), an important distinction from our study, where we include later time points that encompass the peak of branching (DIV 10-12). At DIV 12, dendrite branching was altered by stiffness for both pure and mixed neuronal cultures. Furthermore, we treated hippocampal cultures with glutamate receptor antagonists and with astrocyte-conditioned media. Blocking AMPA and NMDA receptors affected the changes in dendrite branching seen at varying rigidities. Moreover, extracellular factors secreted by astrocytes also change dendrite branching seen at varying rigidities. Thus, astrocytes and ionotropic glutamate receptors contribute to mechanosensing.

Effects of substrate stiffness and cell density on primary hippocampal cultures.

Previous studies have shown that dendrites are influenced by substrate stiffness when neurons are plated in either pure or mixed cultures. However, because substrate rigidity can also affect other aspects of culture development known to impact dendrite branching, such as overall cell number, it is unclear whether substrate stiffness exerts a direct or indirect effect on dendrite morphology. In this study, we determine whether substrate stiffness plays a critical role in regulating dendrite branching independent of cell number. We plated primary mixed hippocampal cultures on soft and stiff gels, with Young's moduli of 1 kPa and 7 kPa, respectively. We found that neurons plated on stiffer substrates showed increased branching relative to neurons grown on softer substrates at the same cell number. On the stiff gels, we also observed a cell number-dependent effect, in which increasing initial plating density decreased dendrite branching. This change correlates with an increase in extracellular glutamate. We concluded that both cell number and substrate stiffness play roles in determining dendrite branching, and that the two effects are independent of one another.

Identification and quantification of skeletal myotube contraction and association in vitro by video microscopy.

Skeletal muscle is the largest tissue in the body by weight and plays many roles in maintaining homeostasis and health. Ex vivo cell-based experimental systems used to study muscle cell contraction, and others based on incorporation of cells into sensitive force transducers or electrophysiology equipment, are time-consuming, invasive, and not universally available, slowing the pace of research. Video microscopy provides a noninvasive way to record the contractile behavior of skeletal muscle cells in vitro. We have developed a numerical procedure using image processing and pattern recognition algorithms, that makes it possible to quantify contractile behavior of multiple myotubes simultaneously, based on video data. We examined the ability of the program to identify movement using a simplified graphical model of myotube contraction and found that the program's success is dependent on the morphology and movement characteristics of the objects. However, the program performs optimally over the types of motions approximating those observed in culture and identifies contracting myotubes in sample videomicrographs of muscle cells in vitro. This program quantifies contractility on a population level, can be adapted for use in laboratories capable of digital video capture from a microscope, and may be coupled with other experimental techniques to supplement existing research tools.

Medical student research exposure via a series of modular research programs.

BACKGROUND: The falling percentage of doctors of medicine applying for National Institute of Health-funded research grants is 1 indicator that physician-scientists are a disappearing breed. This is occurring at a time when increased translational, disease-oriented, patient-oriented, and clinical research are national goals. One of the keys to providing sufficient numbers of physician-scientists to support this goal is the active targeting of medical students. We hypothesize that an improved research program infrastructure and responsiveness to changing student needs will increase student participation in research-oriented electives. METHODS: We have developed a student research program consisting of 2 Students Interested in Research noncredit electives (lecture and laboratory based), summer fellowships, support for year-out fellowships, and a Distinction in Research program that spans undergraduate medical education. Student participation and short-term research outcomes from fall 2004 through spring 2008 are analyzed to examine program efficacy. RESULTS: Students involved in the early parts of the program initially experienced higher application and success rates for summer funding opportunities, but as the program has matured, these rates have fallen in line with the class average. Independently, students participating in later portions of the program increasingly submit or publish a first author paper and have taken a year off for research during medical school. Overlap of participation in the programs is generally smaller than expected. CONCLUSION: Although structured programs can provide step-wise research experiences of increasing intensity, students may not experience a training pipeline in which each stage relies on those before and after, and instead may sample an a la carte selection of research-based enrichment opportunities.