eScholarship@UMMS

STUDY DESIGN: A systematic review of the available medical literature from 1980 to 2010 was conducted and combined with expert opinion from a recent survey of experts regarding cervical spine fractures. Using an objective, hierarchical approach, the best available evidence is presented for health-related quality-of-life outcomes for these injuries.

OBJECTIVE: To provide an evidence-based set of guidelines for cervical spine injuries in order to reduce variability in the information given to patients and their families.

SUMMARY OF BACKGROUND DATA: Patients' expectations regarding quality-of-life outcomes are highly dependent on the information provided by surgeons early in the treatment course. Our previous work has demonstrated that there is substantial variability in what surgeons tell patients regarding outcomes of cervical spine injuries, thus patients' expectations will differ and outcomes vary.

METHODS: Four common cervical spine injuries (C1 burst, Hangman fracture, odontoid fracture, and unilateral facet fracture) treated both surgically and nonsurgically were considered. We assessed the evidence regarding 5 health-related quality-of-life outcomes: time to return to work, activity level, hospital stay, the proportion of patients who are pain free and patients who have regained full range of motion at 1 year after the injury.

RESULTS: Published outcome data were available for most injuries. Using consensus expert opinion and the literature, answers to each question were achieved. Overall, expert opinion was relatively homogeneous across injury types, suggesting that experts do not distinguish between specific injuries when advising patients of expected outcomes such as pain.

CONCLUSION: By overcoming gaps in the literature with consensus expert opinion, our study provides surgeons and others with evidence-based medicine guidelines for patient-centered outcomes after cervical spine injury. This information can be presented to patients to frame expectations of typical outcomes during and after treatment to optimize patient care and quality of life.

Unicameral bone cysts are rare in adults and are most often found incidentally on radiographs. However, they can persist from the adolescent period and may be present in locations that predispose to or exacerbate fractures.This article describes a case of a healthy 40-year-old woman who sustained a proximal humerus trauma that involved a large unicameral bone cyst, resulting in a 3-part head-splitting fracture. The epiphyseal location of the cyst contributed to the severity and extent of the fracture that resulted from a simple fall. Given the age of the patient, open reduction and internal fixation with a locking plate and lag screws was performed. The patient chose open reduction and internal fixation to preserve a hemiarthroplasty procedure in case of future revision. Successful humeral head reconstruction was achieved, and the patient fully recovered. One year postoperatively, the patient underwent arthroscopic debridement to alleviate subjective stiffness and decreased range of motion.Multipart head-splitting fractures require complex repair strategies. The gold standard for the treatment of these injuries is hemiarthroplasty. However, the decision process is difficult in a young patient given the average survival of autoplastic prostheses and the added difficulty of later revision. The current case demonstrates the complexity of decision making resulting from a rare injury in a young, healthy patient and shows that open reduction and internal fixation can provide acceptable reconstruction in such situations.

To present a case report of a patient with an ASIA B spinal cord injury with partially intact baseline IONM who made a complete functional recovery postoperatively. A thirty-three year old male presented after a motor vehicle accident. Imaging studies revealed a C4-C5 bilateral facet dislocation. On presentation the patient had 4/5 strength in bilateral biceps and wrist extensors, 3/5 strength in bilateral triceps, and 0/5 strength in the finger flexors, intrinsics and all lower extremity muscles. Motor level was C7. Sensation was grossly intact to light touch throughout all extremities, intact to pinprick from C2 to T7, and absent to pinprick caudal to T7. Rectal tone and contraction were absent. After attempts at closed reduction failed the patient underwent an open reduction and posterior C4-C5 fusion. Intraoperative neurophysiologic monitoring (IONM) revealed the presence of baseline responses to the posterior tibial nerve using somatosensory evoked potentials and to the right abductor hallucis using transcranial motor evoked potentials. At the 6 weeks postoperative visit the patient had full 5/5 motor strength to all muscles except the left deltoid that was 4/5 due to a rotator cuff injury. This case illustrates a potential prognostic value of IONM. Despite lack of clinical motor function at the time of surgery, IONM was able to illicit a motor response in the right lower extremity. Further prospective studies are needed for further investigation.

BACKGROUND: C5 nerve palsy is a known complication of cervical spine surgery. The development and etiology of this complication are not completely understood. The purpose of the present study was to determine whether rotation of the cervical spinal cord predicts the development of a C5 palsy.

METHODS: We performed a retrospective review of prospectively collected spine registry data as well as magnetic resonance images. We reviewed the records for 176 patients with degenerative disorders of the cervical spine who underwent anterior cervical decompression or corpectomy within the C4 to C6 levels. Our measurements included area for the spinal cord, space available for the cord, and rotation of the cord with respect to the vertebral body.

RESULTS: There was a 6.8% prevalence of postoperative C5 nerve palsy as defined by deltoid motor strength of /= 11 degrees ) and palsy (point-biserial correlation = 0.94; p < 0.001). A diagnostic criterion of 6 degrees of rotation could identify patients who had a C5 palsy (sensitivity = 1.00 [95% confidence interval, 0.70 to 1.00], specificity = 0.97 [95% confidence interval, 0.93 to 0.99], positive predictive value = 0.71 [95% confidence interval, 0.44 to 0.89], negative predictive value = 1.00 [95% confidence interval, 0.97 to 1.00]).

CONCLUSIONS: Our evidence suggests that spinal cord rotation is a strong and significant predictor of C5 palsy postoperatively. Patients can be classified into three types, with Type 1 representing mild rotation (0 degrees to 5 degrees ), Type 2 representing moderate rotation (6 degrees to 10 degrees ), and Type 3 representing severe rotation (>/= 11 degrees ). The rate of C5 palsy was zero of 159 in the Type-1 group, eight of thirteen in the Type-2 group, and four of four in the Type-3 group. This information may be valuable for surgeons and patients considering anterior surgery in the C4 to C6 levels.

STUDY DESIGN: Observational.

OBJECTIVE: To estimate the radiation dose imparted to patients during typical thoracolumbar spinal surgical scenarios.

SUMMARY OF BACKGROUND DATA: Minimally invasive techniques continue to become more common in spine surgery. Computer-assisted navigation systems coupled with intraoperative cone-beam computed tomography (CT) represent one such method used to aid in instrumented spinal procedures. Some studies indicate that cone-beam CT technology delivers a relatively low dose of radiation to patients compared with other x-ray-based imaging modalities. The goal of this study was to estimate the radiation exposure to the patient imparted during typical posterior thoracolumbar instrumented spinal procedures, using intraoperative cone-beam CT and to place these values in the context of standard CT doses.

METHODS: Cone-beam CT scans were obtained using Medtronic O-arm (Medtronic, Minneapolis, MN). Thermoluminescence dosimeters were placed in a linear array on a foam-plastic thoracolumbar spine model centered above the radiation source for O-arm presets of lumbar scans for small or large patients. In-air dosimeter measurements were converted to skin surface measurements, using published conversion factors. Dose-length product was calculated from these values. Effective dose was estimated using published effective dose to dose-length product conversion factors.

RESULTS: Calculated dosages for many full-length procedures using the small-patient setting fell within the range of published effective doses of abdominal CT scans (1-31 mSv). Calculated dosages for many full-length procedures using the large-patient setting fell within the range of published effective doses of abdominal CT scans when the number of scans did not exceed 3.

CONCLUSION: We have demonstrated that single cone-beam CT scans and most full-length posterior instrumented spinal procedures using O-arm in standard mode would likely impart a radiation dose within the range of those imparted by a single standard CT scan of the abdomen. Radiation dose increases with patient size, and the radiation dose received by larger patients as a result of more than 3 O-arm scans in standard mode may exceed the dose received during standard CT of the abdomen. Understanding radiation imparted to patients by cone-beam CT is important for assessing risks and benefits of this technology, especially when spinal surgical procedures require multiple intraoperative scans.

Hematopoietic stem cells (HSCs) have the ability to self-renew and replenish the blood and immune system for the life span of an individual. An age-associated decline in HSC function is responsible for the decreased immune function and increased incidence of myeloid diseases and anemia in the elderly. The changes in HSC function are thought to occur as the result of an intrinsic defect in the self-renewal potential of HSCs as they age. In this chapter, we describe a bone marrow serial transplantation protocol designed to test the self-renewal capacity of HSCs in vivo.

Virtually all motile eukaryotic cilia and flagella have a '9+2' axoneme in which nine doublet microtubules surround two singlet microtubules. Associated with the central pair of microtubules are protein complexes that form at least seven biochemically and structurally distinct central pair projections. Analysis of mutants lacking specific projections has indicated that each may play a unique role in the control of flagellar motility. One of these is the C1d projection previously shown to contain the proteins FAP54, FAP46, FAP74 and FAP221/Pcdp1, which exhibits Ca(2+)-sensitive calmodulin binding. Here we report the isolation and characterization of a Chlamydomonas reinhardtii null mutant for FAP46. This mutant, fap46-1, lacks the C1d projection and has impaired motility, confirming the importance of this projection for normal flagellar movement. Those cells that are motile have severe defects in phototaxis and the photoshock response, underscoring a role for the C1d projection in Ca(2+)-mediated flagellar behavior. The data also reveal for the first time that the C1d projection is involved in the control of interdoublet sliding velocity. Our studies further identify a novel C1d subunit that we term C1d-87, give new insight into relationships between the C1d subunits, and provide evidence for multiple sites of calmodulin interaction within the C1d projection. These results represent significant advances in our understanding of an important but little studied axonemal structure.

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34 degrees C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34 degrees C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.

Cilia and flagella are microtubule-based organelles that protrude from the cell body. Ciliary assembly requires intraflagellar transport (IFT), a motile system that delivers cargo from the cell body to the flagellar tip for assembly. The process controlling injections of IFT proteins into the flagellar compartment is, therefore, crucial to ciliogenesis. Extensive biochemical and genetic analyses have determined the molecular machinery of IFT, but these studies do not explain what regulates IFT injection rate. Here, we provide evidence that IFT injections result from avalanche-like releases of accumulated IFT material at the flagellar base and that the key regulated feature of length control is the recruitment of IFT material to the flagellar base. We used total internal reflection fluorescence microscopy of IFT proteins in live cells to quantify the size and frequency of injections over time. The injection dynamics reveal a power-law tailed distribution of injection event sizes and a negative correlation between injection size and frequency, as well as rich behaviors such as quasiperiodicity, bursting, and long-memory effects tied to the size of the localized load of IFT material awaiting injection at the flagellar base, collectively indicating that IFT injection dynamics result from avalanche-like behavior. Computational models based on avalanching recapitulate observed IFT dynamics, and we further show that the flagellar Ras-related nuclear protein (Ran) guanosine 5'-triphosphate (GTP) gradient can in theory act as a flagellar length sensor to regulate this localized accumulation of IFT. These results demonstrate that a self-organizing, physical mechanism can control a biochemically complex intracellular transport pathway.

Mother-daughter centriole disengagement, the necessary first step in centriole duplication, involves Plk1 activity in early mitosis and separase activity after APC/C activity mediates securin degradation. Plk1 activity is thought to be essential and sufficient for centriole disengagement with separase activity playing a supporting but non-essential role. In separase null cells, however, centriole disengagement is substantially delayed. The ability of APC/C activity alone to mediate centriole disengagement has not been directly tested. We investigate the interrelationship between Plk1 and APC/C activities in disengaging centrioles in S or G2 HeLa and RPE1 cells, cell types that do not reduplicate centrioles when arrested in S phase. Knockdown of the interphase APC/C inhibitor Emi1 leads to centriole disengagement and reduplication of the mother centrioles, though this is slow. Strong inhibition of Plk1 activity, if any, during S does not block centriole disengagement and mother centriole reduplication in Emi1 depleted cells. Centriole disengagement depends on APC/C-Cdh1 activity, not APC/C-Cdc20 activity. Also, Plk1 and APC/C-Cdh1 activities can independently promote centriole disengagement in G2 arrested cells. Thus, Plk1 and APC/C-Cdh1 activities are independent but slow pathways for centriole disengagement. By having two slow mechanisms for disengagement working together, the cell ensures that centrioles will not prematurely separate in late G2 or early mitosis, thereby risking multipolar spindle assembly, but rather disengage in a timely fashion only late in mitosis.

Vomeronasal sensory neurons (VSNs) extend axons to the accessory olfactory bulb (AOB) where they form synaptic connections that relay pheromone signals to the brain. The projections of apical and basal VSNs segregate in the AOB into anterior (aAOB) and posterior (pAOB) compartments. Although some aspects of this organization exhibit fundamental similarities with the main olfactory system, the mechanisms that regulate mammalian vomeronasal targeting are not as well understood. In the olfactory epithelium (OE), the glycosyltransferase beta3GnT2 maintains expression of axon guidance cues required for proper glomerular positioning and neuronal survival. We show here that beta3GnT2 also regulates guidance and adhesion molecule expression in the vomeronasal system in ways that are partially distinct from the OE. In wildtype mice, ephrinA5(+) axons project to stereotypic subdomains in both the aAOB and pAOB compartments. This pattern is dramatically altered in beta3GnT2(-/-) mice, where ephrinA5 is upregulated exclusively on aAOB axons. Despite this, apical and basal VSN projections remain strictly segregated in the null AOB, although some V2r1b axons that normally project to the pAOB inappropriately innervate the anterior compartment. These fibers appear to arise from ectopic expression of V2r1b receptors in a subset of apical VSNs. The homotypic adhesion molecules Kirrel2 and OCAM that facilitate axon segregation and glomerular compartmentalization in the main olfactory bulb are ablated in the beta3GnT2(-/-) aAOB. This loss is accompanied by a two-fold increase in the total number of V2r1b glomeruli and a failure to form morphologically distinct glomeruli in the anterior compartment. These results identify a novel function for beta3GnT2 glycosylation in maintaining expression of layer-specific vomeronasal receptors, as well as adhesion molecules required for proper AOB glomerular formation.

The bacterial lacZ gene is widely used as a reporter in a myriad of mouse transgenic experiments. beta-Galactosidase, encoded by lacZ, is usually detected using X-gal in combination with ferric and ferrous ions. This assay produces a blue indole precipitate that is easy to detect visually. Here, we show that Salmon-gal in combination with tetrazolium salts provides a more sensitive and faster staining reaction than the traditional beta-galactosidase assay in mouse embryos. Using a combination of Salmon-gal and tetranitroblue tetrazolium, we were able to visualize the activity of beta-galactosidase in embryos at stages when the customary X-gal reaction failed to detect staining. Our studies provide an enhanced alternative for beta-galactosidase detection in expression and cell fate studies that use lacZ-based transgenic mouse lines.

UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5' end of a nuclear mRNA, but are also reported to associate with the Exon Junction Complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly we used Fluorescence Recovery after Photobleaching (FRAP) to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the PI3 kinase / AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 Kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intron-less Histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the Endoplasmic Reticulum or to Mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and can promote the nuclear retention of some mRNAs.

Integrin-based adhesions promote cell survival as well as cell motility and invasion. We show here that the adhesion regulatory protein supervillin increases cell survival by decreasing levels of the tumor suppressor protein p53 and downstream target genes. RNAi-mediated knockdown of a new splice form of supervillin (isoform 4) or both isoforms 1 and 4 increases the amount of p53 and cell death, whereas p53 levels decrease after overexpression of either supervillin isoform. Cellular responses to DNA damage induced by etoposide or doxorubicin include down-regulation of endogenous supervillin coincident with increases in p53. In DNA-damaged supervillin knockdown cells, p53 knockdown or inhibition partially rescues the loss of cell metabolic activity, a measure of cell proliferation. Knockdown of the p53 deubiquitinating enzyme USP7/HAUSP also reverses the supervillin phenotype, blocking the increase in p53 levels seen after supervillin knockdown and accentuating the decrease in p53 levels triggered by supervillin overexpression. Conversely, supervillin overexpression decreases the association of USP7 and p53 and attenuates USP7-mediated p53 deubiquitination. USP7 binds directly to the supervillin N terminus and can deubiquitinate and stabilize supervillin. Supervillin also is stabilized by derivatization with the ubiquitin-like protein SUMO1. These results show that supervillin regulates cell survival through control of p53 levels and suggest that supervillin and its interaction partners at sites of cell-substrate adhesion constitute a locus for cross-talk between survival signaling and cell motility pathways.

BACKGROUND: Exposure to dioxins has been associated with delayed pubertal onset in both epidemiologic and animal studies. Whether genetic polymorphisms may modify this association is currently unknown. Identifying such genes could provide insight into mechanistic pathways. This is one of the first studies to assess genetic susceptibility to dioxins.

OBJECTIVES: We evaluated whether common polymorphisms in genes affecting either molecular responses to dioxin exposure or pubertal onset influence the association between peripubertal serum dioxin concentration and male pubertal onset.

METHODS: In this prospective cohort of Russian adolescent boys (n = 392), we assessed gene-environment interactions for 337 tagging single-nucleotide polymorphisms (SNPs) from 46 candidate genes and two intergenic regions. Dioxins were measured in the boys' serum at age 8-9 years. Pubertal onset was based on testicular volume and on genitalia staging. Statistical approaches for controlling for multiple testing were used, both with and without prescreening for marginal genetic associations.

RESULTS: After accounting for multiple testing, two tag SNPs in the glucocorticoid receptor (GR/NR3C1) gene and one in the estrogen receptor-alpha (ESR1) gene were significant (q < 0.2) modifiers of the association between peripubertal serum dioxin concentration and male pubertal onset defined by genitalia staging, although not by testicular volume. The results were sensitive to whether multiple comparison adjustment was applied to all gene-environment tests or only to those with marginal genetic associations.

CONCLUSIONS: Common genetic polymorphisms in the glucocorticoid receptor and estrogen receptor-alpha genes may modify the association between peripubertal serum dioxin concentration and pubertal onset. Further studies are warranted to confirm these findings.

Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results.

Changes in nuclear morphology occur during normal development and have been observed during the progression of several diseases. The shape of a nucleus is governed by the balance of forces exerted by nuclear-cytoskeletal contacts and internal forces created by the structure of the chromatin and nuclear envelope. However, factors that regulate the balance of these forces and determine nuclear shape are poorly understood. The SWI/SNF chromatin remodeling enzyme ATPase, BRG1, has been shown to contribute to the regulation of overall cell size and shape. Here we document that immortalized mammary epithelial cells show BRG1-dependent nuclear shape changes. Specifically, knockdown of BRG1 induced grooves in the nuclear periphery that could be documented by cytological and ultrastructural methods. To test the hypothesis that the observed changes in nuclear morphology resulted from altered tension exerted by the cytoskeleton, we disrupted the major cytoskeletal networks and quantified the frequency of BRG1-dependent changes in nuclear morphology. The results demonstrated that disruption of cytoskeletal networks did not change the frequency of BRG1-induced nuclear shape changes. These findings suggest that BRG1 mediates control of nuclear shape by internal nuclear mechanisms that likely control chromatin dynamics.

Mild age-related declines in visual function occur in humans and monkeys, independent of ocular pathology, suggesting involvement of central visual pathways (Spear [1993] Vision Res 33:2589-2609). Although many factors might account for this decline, a loss of neurons in primary visual cortex (V1) could be a contributing factor. Previous studies of neuron numbers in V1 reported stability across age, but were limited in the ages and genders studied and sampled only limited parts of V1 or limited cell types, allowing for the possibility of a subtle loss of neurons. We pursued this question in 26 behaviorally tested adult male and female rhesus monkeys ranging from 7.4 to 31.0 years of age by using design-based stereology to estimate numbers of NeuN-labeled neurons and thionin-stained glia within three laminar zones, supragranular (layers II-IVB), granular (IVC), and infragranular (V-VI), across the entirety of V1. There were no significant differences between males and females on any measures, except for total brain weight (P = 0.0038). There was an average of 416,000,000 neurons in V1, but no effect of age on this total or numbers within any laminar zone. Similarly, there was an average of 184,000,000 glia in V1 (44% of the number of neurons), but no effect of age on this total. However, there was a significant age-related increase in numbers of glia in the infragranular zone, perhaps reflecting a glial response to pathology in myelinated projection fibers. This study provides further evidence that in normal aging neurons are not lost and hence cannot account for age-related dysfunction.

We have previously shown that myelin abnormalities characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. Although the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. We found significant effects of Klotho on oligodendrocyte functions, including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein analysis indicated that Klotho's downstream effects involve Akt and ERK signal pathways. Klotho increased OPC maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knock-out mice and control littermates revealed that knock-out mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knock-out mice. Strikingly, at the ultrastructural level, Klotho knock-out mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. To decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes and promote repair in multiple sclerosis.