Terrence F. Holekamp

Fast Three-Dimensional Fluorescence Imaging of Activity in Neural Populations by Objective-Coupled Planar Illumination Microscopy

Unraveling the functions of the diverse neural types in any local circuit ultimately requires methods to record from most or all of its cells simultaneously. One promising approach to this goal is fluorescence imaging, but existing methods using laser-scanning microscopy (LSM) are severely limited in their ability to resolve rapid phenomena, like neuronal action potentials, over wide fields. Here we present a microscope that rapidly sections a three-dimensional volume using a thin illumination sheet whose position is rigidly coupled to the objective and aligned with its focal plane. We demonstrate that this approach allows exceptionally low-noise imaging of large neuronal populations at pixel rates at least 100-fold higher than with LSM. Using this microscope, we studied the pheromone-sensing neurons of the mouse vomeronasal organ and found that responses to dilute urine are largely or exclusively restricted to cells in the apical layer, the location of V1r-family-expressing neurons.

Sulfated Steroids as Natural Ligands of Mouse Pheromone-Sensing Neurons

Among mice, pheromones and other social odor cues convey information about sex, social status, and identity; however, the molecular nature of these cues is essentially unknown. To identify these cues, we screened chromatographic fractions of female mouse urine for their ability to cause reproducible firing rate increases in the pheromone-detecting vomeronasal sensory neurons (VSNs) using multielectrode array (MEA) recording. Active compounds were found to be remarkably homogenous in their basic properties, with most being of low molecular weight, moderate hydrophobicity, low volatility, and possessing a negative electric charge. Purification and structural analysis of active compounds revealed multiple sulfated steroids, of which two were identified as sulfated glucocorticoids, including corticosterone 21-sulfate. Sulfatase-treated urine extracts lost >80% of their activity, indicating that sulfated compounds are the predominant VSN ligands in female mouse urine. As measured by MEA recording, a collection of 31 synthetic sulfated steroids triggered responses 30-fold more frequently than did a similarly sized stimulus set containing the majority of all previously reported VSN ligands. Collectively, VSNs detected all major classes of sulfated steroids, but individual neurons were sensitive to small variations in chemical structure. VSNs from both males and females detected sulfated steroids, but knock-outs for the sensory transduction channel TRPC2 did not detect these compounds. Urine concentrations of the two sulfated glucocorticoids increased many fold in stressed animals, indicating that information about physiological status is encoded by the urine concentration of particular sulfated steroids. These results provide an unprecedented characterization of the signals available for chemical communication among mice.

Intrasite vancomycin powder for the prevention of surgical site infection in spine surgery: a systematic literature review

BACKGROUND CONTEXT Deep surgical site infections (SSIs) following spinal surgery are a significant burden to the patient, patients family, and the health-care system. Because of increasing pressures to reduce SSIs and control costs, some spine surgeons have begun placing lyophilized vancomycin powder directly into the surgical wound at the conclusion of the procedure. However, the literature supporting this practice remains limited. PURPOSE To review the current literature examining the use of prophylactic intrasite vancomycin powder to control SSIs in spinal surgery and determine if any standard recommendations can be made. STUDY DESIGN A systematic review. METHODS Ovid Medline and PubMed were searched to identify English language articles. RESULTS No current guidelines are available for the use of intrasite vancomycin powder in preventing SSIs, and no standard dosage for the drug exists. Based on the limited literature and evidence currently available, there appears to be a protective effect of intrasite vancomycin powder on the incidence of SSI, without evidence of side effects. However, case reports do exist describing the systemic side effects after intrasite vancomycin powder during spine surgery. CONCLUSIONS The interpretation of the available evidence supporting the use of intrasite vancomycin powder in surgical wounds is limited, and its extrapolation should be performed with caution. Despite the lack of significant high-quality evidence available in the literature, many surgeons have adopted this practice; anecdotally, it continues to provide protection from infection without apparent significant risk of side effects.

Structural Characterization of Sulfated Steroids That Activate Mouse Pheromone-Sensing Neurons

In many species, social behavior is organized via chemical signaling. While many of these signals have been identified for insects, the chemical identity of these social cues (often called pheromones) for mammals is largely unknown. We recently isolated these chemical cues that caused firing in the pheromone-sensing neurons of the vomeronasal organ from female mouse urine [Nodari, F., et al. (2008) J. Neurosci. 28, 6407-6418]. Here, we report their structural characterization. Mass spectrometric approaches, including tandem quadrupole, multiple-stage linear ion trap, high-resolution mass spectrometry, and H-D exchange followed by ESI mass spectrometry, along with (1)H and (13)C nuclear magnetic resonance spectroscopy, including two-dimensional correlation spectroscopy, total correlation spectroscopy, heteronuclear multiple-quantum coherence, and NOE, were used to identify two sulfated steroids, 4-pregnene-11beta,20,21-triol-3-one 21-sulfate (I) (the configuration at C20 was not deduced) and 4-pregnene-11beta,21-diol-3,20-dione 21-sulfate (II), whose presence is sex-specific. The identification of this novel class of mammalian social signaling compounds suggests that steroid hormones, upon conjugation, assume a new biological role, conveying information about the organisms identity and physiological state.

Relationship of syrinx size and tonsillar descent to spinal deformity in Chiari malformation Type I with associated syringomyelia

OBJECT Chiari malformation Type I (CM-I) is a developmental abnormality often associated with a spinal syrinx. Patients with syringomyelia are known to have an increased risk of scoliosis, yet the influence of specific radiographically demonstrated features on the prevalence of scoliosis remains unclear. The primary objective of the present study was to investigate the relationship of maximum syrinx diameter and tonsillar descent to the presence of scoliosis in patients with CM-I-associated syringomyelia. A secondary objective was to explore the role of craniovertebral junction (CVJ) characteristics as additional risk factors for scoliosis. METHODS The authors conducted a retrospective review of pediatric patients evaluated for CM-I with syringomyelia at a single institution in the period from 2000 to 2012. Syrinx morphology and CVJ parameters were evaluated with MRI, whereas the presence of scoliosis was determined using standard radiographic criteria. Multiple logistic regression was used to analyze radiological features that were independently associated with scoliosis. RESULTS Ninety-two patients with CM-I and syringomyelia were identified. The mean age was 10.5 ± 5 years. Thirty-five (38%) of 92 patients had spine deformity; 23 (66%) of these 35 patients were referred primarily for deformity, and 12 (34%) were diagnosed with deformity during workup for other symptoms. Multiple regression analysis revealed maximum syrinx diameter > 6 mm (OR 12.1, 95% CI 3.63-40.57, p < 0.001) and moderate (5-12 mm) rather than severe (> 12 mm) tonsillar herniation (OR 7.64, 95% CI 2.3-25.31, p = 0.001) as significant predictors of spine deformity when controlling for age, sex, and syrinx location. CONCLUSIONS The current study further elucidates the association between CM-I and spinal deformity by defining specific radiographic characteristics associated with the presence of scoliosis. Specifically, patients presenting with larger maximum syrinx diameters (> 6 mm) have an increased risk of scoliosis.

Delayed synostoses of uninvolved sutures after surgical treatment of nonsyndromic craniosynostosis

BackgroundCraniosynostosis causes significant cranial deformity in the pediatric population. Open and endoscopic-assisted surgeries have led to increasingly successful management of this condition. Following surgical reconstruction, subsequent development of postnatal synostosis of previously patent sutures have been described and noted to be most frequently associated with multisuture synostosis patients with syndromic diagnoses. Very rarely, postsurgical new sutural fusion has been identified in nonsyndromic patients who initially present with isolated single-suture synostosis. The purpose of this study was to evaluate the incidence of new synostosis among patients who had undergone craniosynostosis reconstruction with either the open or endoscopic technique. MethodsPatients undergoing open and endoscopic surgery for nonsyndromic craniosynostosis were reviewed. Preoperative and postoperative imaging and clinical information were reviewed, and cases showing progressive cranial deformity were identified. ResultsThree (2.1%) of 145 patients undergoing open craniosynostosis surgery and 2 (1.7%) of 121 patients undergoing endoscopic surgery developed delayed fusion of an additional suture during follow-up. This was identified at a median of 16.4 months after initial surgery in the open group and 15.25 months after surgery in the endoscopic group. In patients undergoing open surgery, each patient developed new sagittal synostosis after initial presentation of coronal synostosis in 1 patient and metopic synostosis in 2 patients. In patients undergoing endoscopic surgery, each patient developed new coronal synostosis after sagittal repair. ConclusionsManagement of craniosynostosis has evolved over time with increasing availability of effective and safe treatments. During long-term follow-up, a small number of patients may develop premature closure of a different suture that did not undergo surgical manipulation. In our case, series, we identified 3 patients undergoing open surgery and 2 patients undergoing endoscopic surgery for nonsyndromic, single-suture craniosynostosis. This finding supports the necessity of long-term clinical follow-up and the utility of delayed imaging when clinical suspicion indicates.

Circumferential vertebrectomy with reconstruction for holocervical aneurysmal bone cyst at C4 in a 15-year-old girl.

Study Design. The authors describe the unique case of a 15-year-old girl with a holovertebral aneurysmal bone cyst at C4, causing anterolisthesis and kyphosis, who underwent circumferential vertebrectomy with reconstruction and rigid fusion. Objective. To report an unusual manifestation of aneurysmal bone cyst, requiring special considerations for surgery. The discussion highlights emerging principles for treatment of this lesion, and cervical spine fusion, in the pediatric population. Summary of Background Data. There is no previous report of a holovertebral aneurysmal bone cyst of the pediatric cervical spine. The potential for neurologic or vascular compromise from this lesion is substantial if left untreated, and the risk of recurrence or other morbidity is significantly higher unless completely resected. Methods. A 15-year-old white female presented with a 4-month history of neck pain after a mild injury, but was neurologically intact. Diagnostic imaging revealed a holovertebral, multicystic, and osteolytic lesion with multiple fluid–fluid levels in the fourth cervical vertebra. Total vertebrectomy and repair were performed with fibular strut grafts and placement of rigid anterior and posterior instrumentation. This was accomplished in a single anteroposterior operative pass. Results. Surgical treatment produced a stable bony fusion with no neurologic or vascular sequelae. This approach minimizes the risk of recurrence and the possibility of postoperative spinal instability. Conclusion. Spinal aneurysmal bone cyst in children presents diverse challenges. These lesions should be treated with complete resection to minimize the chance of recurrence. In pediatric cases, defects created by resection should be corrected by fusion to minimize the risk of postoperative instability and growth abnormality. One-year followup demonstrated a stable construct, and the patient remains neurologically at her baseline.

Risks and outcomes of spinal deformity surgery in Chiari malformation, Type 1, with syringomyelia versus adolescent idiopathic scoliosis.

BACKGROUND CONTEXT Chiari malformation, Type 1, with syringomyelia (CIM+SM) is often associated with spinal deformity. The safety of scoliosis surgery this population is controversial and has never been directly compared with adolescent idiopathic scoliosis (AIS). PURPOSE The purpose of this study was to compare the safety and subjective outcomes of spinal deformity surgery between patients with Chiari malformation Type 1-associated scoliosis and a matched AIS cohort. STUDY DESIGN This study is based on a retrospective matched cohort analysis. PATIENT SAMPLE Patients with CIM+SM and treated with spinal fusion for spinal deformity were identified in the surgical records of a single institution and were matched, 1:1, with AIS patients undergoing spinal fusion at the same institution. OUTCOME MEASURES The outcome measures were neurological monitoring data quality and integrity, radiographic parameters, and Scoliosis Research Society Questionnaire-22 (SRS-22) scores. METHODS A clinical database was reviewed for patients undergoing spinal reconstruction for CIM+SM-associated spinal deformity at our institution from 2000 to 2012. Thirty-six CIM+SM patients were identified and matched to an AIS cohort (1:1) based on age, gender, major curve magnitude, fusion length, and revision status. Demographics, deformity morphology, surgical details, neuromonitoring data, and preoperative and postoperative SRS-22 scores were recorded at a minimum of 2-year follow-up. Changes in SRS-22 scores were compared within and between groups. Complications and neurological monitoring data issues were compared between groups. RESULTS Mean age was 14.5±5 years (CIM+SM: 14.6±5; AIS: 14.4±5), and 42% of patients were male. Preoperative mean major coronal Cobb measured 58°±25° versus 57°±17° (p=.84) with mean kyphosis 52°±17° versus 41°±20° (p=.018). An average of 10.4±2.6 vertebral levels were fused (10.4±2.8 vs. 10.4±2.3, p=.928). No differences existed in surgical approach (p=.336), estimated blood loss (680±720 vs. 660±310 mL, p=.845), or duration of surgery (6.0±2.2 vs. 5.6±2 hours, p=.434). Complication rate was comparable between the two groups (33% vs. 14%, p=.052). Chiari malformation, Type 1, with syringomyelia experienced more neurological complications (11% vs. 0%, p=.04) and neuromonitoring difficulties (28% vs. 3%, p=.007) than the AIS cohort. Mean curve correction was comparable at 2 years (58% CIM+SM vs. 64% AIS, p=.2). At follow-up, both CIM+SM and AIS groups demonstrated improved cumulative SRS-22 outcome subscores (CIM+SM: +0.4, p=.027; AIS: +0.3, p<.001). No difference in outcome subscores existed between CIM+SM and AIS groups. CONCLUSIONS Although CIM+SM patients undergoing spine reconstruction can expect similar deformity corrections and outcome scores to AIS patients, they also experience higher rates of neuromonitoring difficulties and neurological complications related to surgery. Surgeons should be prepared for these difficulties, particularly in children with larger syrinx size.

Complications and outcomes of complex spine reconstructions in poliomyelitis-associated spinal deformities: a single-institution experience.

Study Design. Retrospective case series. Objective. To share our institutional experience with spinal reconstruction for deformity correction in patients with a history of poliomyelitis. Summary of Background Data. Polio and postpolio syndrome are not uncommonly related to a paralytic spinal deformity. Limited modern data exist regarding outcomes and complications after spinal reconstruction in this population. Methods. A clinical database was reviewed for patients undergoing spinal reconstruction for polio-associated spinal deformity at our institution from 1985 to 2012. Relevant demographic, medical, surgical, and postoperative information were collected from medical records and analyzed. Preoperative and last follow-up Scoliosis Research Society-22 Questionnaire scores were recorded. Results. A total of 22 patients with polio who underwent surgical deformity correction were identified. Mean age was 49 years (range, 12–74 yr), and 15 patients (68%) were female. Preoperative motor deficit was present in 14 of 22 (64%) patients. All patients underwent instrumented spinal fusion (mean, 13 vertebral levels, range, 3–18). Ten (10/22, 45%) patients developed major complications, and 4 patients (4/22, 18%) developed new postoperative neurological deficits. Neurological monitoring yielded a 13% false-negative rate. At 2-year follow-up, 20 of 22 patients maintained an average coronal correction of 25° (33%, P = 0.001) and sagittal correction of 25° (34%, P = 0.003). Minimum 2-year follow-up data were available for 11 of 22 (50%) patients. At an average of 72 months of follow-up (range, 28–134 mo), the mean Scoliosis Research Society-22 Questionnaire pain subscore improved from a mean of 2.75 to 3.6 (P = 0.012); self-image from 2.8 to 3.7 (P = 0.041); function from 3.1 to 3.8 (P = 0.036); satisfaction from 2.1 to 3.9 (P = 0.08); and mental health from 3.7 to 4.5 (P = 0.115). Conclusion. Spine reconstruction for poliomyelitis-associated deformity was associated with high complication rates (54%) and sometimes unreliable neurological monitoring data. Despite this, patients undergoing spine reconstructions had significantly improved outcome scores. These data may help surgeons to appropriately counsel this complicated patient population.