Comparative effectiveness of antinociceptive gene therapies in animal models of diabetic neuropathic pain.

Peripheral neuropathic pain is one of the most common and debilitating complications of diabetes. Several genes have been shown to be effective in reducing neuropathic pain in animal models of diabetes after transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)1-based vectors, yet there has never been a comparative analysis of their efficacy. We compared four different HSV1-based vectors engineered to produce one of two opioid receptor agonists (enkephalin or endomorphin), or one of two isoforms of glutamic acid decarboxylase (GAD65 or GAD67), alone and in combination, in the streptozotocin-induced diabetic rat and mouse models. Our results indicate that a single subcutaneous hindpaw inoculation of vectors expressing GAD65 or GAD67 reduced diabetes-induced mechanical allodynia to a degree that was greater than daily injections of gabapentin in rats. Diabetic mice that developed thermal hyperalgesia also responded to GAD65 or endomorphin gene delivery. The results suggest that either GAD65 or GAD67 vectors are the most effective in the treatment of diabetic pain. The vector combinations, GAD67+endomorphin, GAD67+enkephalin or endomorphin+enkephalin also produced a significant antinociceptive effect but the combination did not appear to be superior to single gene treatment. These findings provide further justification for the clinical development of antinociceptive gene therapies for the treatment of diabetic peripheral neuropathies.

MRI evidence that glibenclamide reduces acute lesion expansion in a rat model of spinal cord injury.

STUDY DESIGN: Experimental, controlled, animal study. OBJECTIVES: To use non-invasive magnetic resonance imaging (MRI) to corroborate invasive studies showing progressive expansion of a hemorrhagic lesion during the early hours after spinal cord trauma and to assess the effect of glibenclamide, which blocks Sur1-Trpm4 channels implicated in post-traumatic capillary fragmentation, on lesion expansion. SETTING: Baltimore. METHODS: Adult female Long-Evans rats underwent unilateral impact trauma to the spinal cord at C7, which produced ipsilateral but not contralateral primary hemorrhage. In series 1 (six control rats and six administered glibenclamide), hemorrhagic lesion expansion was characterized using MRI at 1 and 24 h after trauma. In series 2, hemorrhagic lesion size was characterized on coronal tissue sections at 15 min (eight rats) and at 24 h after trauma (eight control rats and eight administered glibenclamide). RESULTS: MRI (T2 hypodensity) showed that lesions expanded 2.3±0.33-fold (P<0.001) during the first 24 h in control rats, but only 1.2±0.07-fold (P>0.05) in glibenclamide-treated rats. Measuring the areas of hemorrhagic contusion on tissue sections at the epicenter showed that lesions expanded 2.2±0.12-fold (P<0.001) during the first 24 h in control rats, but only 1.1±0.05-fold (P>0.05) in glibenclamide-treated rats. Glibenclamide treatment was associated with significantly better neurological function (unilateral BBB scores) at 24 h in both the ipsilateral (median scores, 9 vs 0; P<0.001) and contralateral (median scores, 12 vs 2; P<0.001) hindlimbs. CONCLUSION: MRI is an accurate non-invasive imaging biomarker of lesion expansion and is a sensitive measure of the ability of glibenclamide to reduce lesion expansion.

Ultrafast strain engineering in complex oxide heterostructures.

We report on ultrafast optical experiments in which femtosecond midinfrared radiation is used to excite the lattice of complex oxide heterostructures. By tuning the excitation energy to a vibrational mode of the substrate, a long-lived five-order-of-magnitude increase of the electrical conductivity of NdNiO(3) epitaxial thin films is observed as a structural distortion propagates across the interface. Vibrational excitation, extended here to a wide class of heterostructures and interfaces, may be conducive to new strategies for electronic phase control at THz repetition rates.

Control of gradient-driven instabilities using shear Alfvén beat waves.

A new technique for manipulation and control of gradient-driven instabilities through nonlinear interaction with Alfvén waves in a laboratory plasma is presented. A narrow, field-aligned density depletion is created in the Large Plasma Device, resulting in coherent, unstable fluctuations on the periphery of the depletion. Two independent shear Alfvén waves are launched along the depletion at separate frequencies, creating a nonlinear beat-wave response at or near the frequency of the original instability. When the beat wave has sufficient amplitude, the original unstable mode is suppressed, leaving only the beat-wave response, generally at lower amplitude.

Specific heat measurements of Ba(0.68)K(0.32)Fe2As2 single crystals: evidence for a multiband strong-coupling superconducting state.

The specific heat of high-purity Ba(0.68)K(0.32)Fe2As2 single crystals with the highest reported superconducting Tc=38.5 K was studied. The electronic specific heat Cp below Tc shows two gap features, with Δ1≈11 meV and Δ2≈3.5 meV obtained from an α-model analysis. The reduced gap value, 2Δ(max)/kBTc≈6.6, the magnitude of the specific-heat jump, ΔCp(Tc)/Tc, and its slope below Tc exhibit a strong-coupling character. We also show that an Eliashberg model with two hole and two electron bands gives the correct values of Tc, the superconducting gaps, and the free-energy difference.

Mechanisms and implications of adaptive immune responses after traumatic spinal cord injury.

Traumatic spinal cord injury (SCI) in mammals causes widespread glial activation and recruitment to the CNS of innate (e.g. neutrophils, monocytes) and adaptive (e.g. T and B lymphocytes) immune cells. To date, most studies have sought to understand or manipulate the post-traumatic functions of astrocytes, microglia, neutrophils or monocytes. Significantly less is known about the consequences of SCI-induced lymphocyte activation. Yet, emerging data suggest that T and B cells are activated by SCI and play significant roles in shaping post-traumatic inflammation and downstream cascades of neurodegeneration and repair. Here, we provide neurobiologists with a timely review of the mechanisms and implications of SCI-induced lymphocyte activation, including a discussion of different experimental strategies that have been designed to manipulate lymphocyte function for therapeutic gain.

Robust axonal growth and a blunted macrophage response are associated with impaired functional recovery after spinal cord injury in the MRL/MpJ mouse.

Spinal cord injury (SCI) in mammals leads to a robust inflammatory response followed by the formation of a glial and connective tissue scar that comprises a barrier to axonal regeneration. The inbred MRL/MpJ mouse strain exhibits reduced inflammation after peripheral injury and shows true regeneration without tissue scar formation following an ear punch wound. We hypothesized that following SCI, the unique genetic wound healing traits of this strain would result in reduced glial and connective tissue scar formation, increased axonal growth, and improved functional recovery. Adult MRL/MpJ and C57BL/6J mice were subjected to a mid-thoracic spinal contusion and the distribution of axon profiles and selected cellular and extracellular matrix components was compared at 1, 2, 4 and 6 weeks post-injury. Recovery of hind-limb locomotor function was assessed over the same time period. The MRL/MpJ mice exhibited robust axon growth within the lesion, beginning at 4 weeks post-injury. This growth was accompanied by reduced macrophage staining at 1, 2, 4 and 6 weeks post-injury, decreased chondroitin sulfate proteoglycan staining at 1-2 weeks and increased laminin staining throughout the lesion at 2-6 weeks post-injury. Paradoxically, the extent of locomotor recovery was impaired in the MRL/MpJ mice. Close examination of the chronic lesion site revealed evidence of ongoing degeneration both within and surrounding the lesion site. Thus, the regenerative genetic wound healing traits of the MRL/MpJ mice contribute to the evolution of a lesion environment that supports enhanced axon growth after SCI. However, this response occurs at the expense of meaningful functional recovery.

Inflammatory-mediated injury and repair in the traumatically injured spinal cord.

Spinal cord trauma activates the immune system and elicits leukocyte recruitment to the site of injury. This increase in immunological activity contributes to acute lesion expansion over a period of days to weeks following the initial trauma. At the same time, inflammatory cells and mediators facilitate endogenous repair processes such as axonal sprouting and remyelination. Thus, to be effective, therapies that target the immune system must limit the destructive effects of neutrophil, macrophage and lymphocyte activation, while simultaneously preserving their reparative functions.

Bone marrow chimeric rats reveal the unique distribution of resident and recruited macrophages in the contused rat spinal cord.

Brain and spinal cord inflammation that develops after traumatic injury is believed to differentially influence the structural and/or physiological integrity of surviving neurons and glia. It is possible that the functional dichotomy of CNS inflammation results from the activity of a heterogeneous macrophage population elicited by trauma. Indeed, unique functions have been attributed to macrophages derived from resident microglia versus those originating from infiltrating monocytes. Thus, whether progressive tissue injury or repair is favored could be explained by the disproportionate contributions of one macrophage subset relative to the other. Descriptive neuroanatomical studies are a reasonable first approach to revealing a relationship between microglia, recruited blood monocytes/macrophages, and regions of tissue degeneration and/or repair. Unfortunately, it is not possible to differentiate between CNS macrophage subsets using conventional immunohistochemical approaches. In the present study, we have used radiation bone marrow chimeric rats to definitively characterize the macrophage reaction elicited by experimental spinal contusion injury. In chimeric animals, antibodies raised against unique cell surface molecules expressed on bone marrow-derived cells (BMCs) were used to distinguish infiltrating BMCs from resident microglial-derived macrophages. Our findings indicate that the onset and plateau of macrophage activation (previously shown to be 3 and 7 days postinjury, respectively) is dominated initially by microglial-derived macrophages and then is supplanted by hematogenous cells. While resident macrophages are ubiquitously distributed throughout the injury site, leukocyte-derived monocytes exclusively infiltrate the gray matter and to a lesser extent subpial white matter. Generally, monocyte foci in white matter remain associated with the lumen or abluminal surface of blood vessels, i.e. few cells actually infiltrate the parenchyma. If functional differences exist between CNS macrophage subsets, differences in the time-dependent accumulation and distribution of these cell types could differentially influence the survival of surrounding neurons and glia.

Spinal cord neuropathology in rat experimental autoimmune encephalomyelitis: modulation by oral administration of myelin basic protein.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) in which clinical neurological signs and histopathologic changes of disease can be suppressed by feeding CNS myelin proteins. Using immunohistochemistry and image analysis, the cellular immune response was quantified over the rostral-caudal axis of the spinal cord in rats with EAE and in animals fed high- or low-dose myelin basic protein (MBP) prior to inducing EAE (tolerized animals). In a subset of rats, MBP was fed 9 days after MBP immunization to examine the effect of oral tolerance on the progression of CNS pathology. In unfed rats or rats fed vehicle only, activated microglia and macrophages were co-localized with T-lymphocytes throughout the spinal cord, but greater cellular reactions were evident in gray matter relative to white matter. In all tolerized animals, the CNS inflammatory response was reduced relative to controls. Subtle pathologic changes were occasionally observed in the CNS of MBP-fed animals, but the distribution of inflammatory cells in the dorso-ventral axis was more polarized in animals fed high-dose MBP. In this group, more T-cells and activated microglia were present in the dorsal spinal cord, specifically in the gray matter. In the group fed MBP after disease induction, clinical disease progressed as in control non-fed rats, but recovery from disease appeared to be accelerated. Thus, the results presented here provide a comprehensive analysis of the distribution and magnitude of inflammatory cells within the spinal cord in EAE and challenge the theory that MBP-induced EAE is only a white matter disease. These data also describe how the activation and distribution of immune effector cells is altered by oral tolerance and may help predict a range of neurological deficits not previously appreciated in EAE, particularly those effected by gray matter pathology.

The neuropathological and behavioral consequences of intraspinal microglial/macrophage activation.

Activated microglia and macrophages (CNS macrophages) have been implicated in the secondary or "bystander" pathology (e.g. axon injury, demyelination) that accompanies traumatic or autoimmune injury to the brain and spinal cord. These cells also can provide neurotrophic support and promote axonal regeneration. Studying the divergent functional potential of CNS macrophages in trauma models is especially difficult due to the various degradative mechanisms that are initiated prior to or concomitant with microglial/macrophage activation (e.g. hemorrhage, edema, excitotoxicity, lipid peroxidation). To study the potential impact of activated CNS macrophages on the spinal cord parenchyma, we have characterized an in vivo model of non-traumatic spinal cord neuroinflammation. Specifically, focal activation of CNS macrophages was achieved using stereotaxic microinjections of zymosan. Although microinjection does not cause direct mechanical trauma, localized activation of macrophages with zymosan acts as an "inflammatory scalpel" causing tissue injury at and nearby the injection site. The present data reveal that activation of CNS macrophages in vivo can result in permanent axonal injury and demyelination. Moreover, the pathology can be graded and localized to specific white matter tracts to produce quantifiable behavioral deficits. Further development of this model will help to clarify the biological potential of microglia and macrophages and the molecular signals that control their function within the spinal cord.

Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats.

The distribution of microglia, macrophages, T-lymphocytes, and astrocytes was characterized throughout a spinal contusion lesion in Sprague-Dawley and Lewis rats by using immunohistochemistry. The morphology, spatial localization, and activation state of these inflammatory cells were described both qualitatively and quantitatively at 12 hours, 3, 7, 14, and 28 days after injury. By use of OX42 and ED1 antibodies, peak microglial activation was observed within the lesion epicenter of both rat strains between three and seven days post-injury preceding the bulk of monocyte influx and macrophage activation (seven days). Rostral and caudal to the injury site, microglial activation plateaued between two and four weeks post-injury in the dorsal and lateral funiculi as indicated by morphological transformation and the de-novo expression of major histocompatibility class II (MHC II) molecules. Similar to the timing of microglial reactions, T-lymphocytes maximally infiltrated the lesion epicenter between three and seven days post-injury. Reactive astrocytes, while present in the acute lesion, were more prominent at later survival times (7-28 days). These cells were interspersed with activated microglia but appeared to surround and enclose tissue sites occupied by reactive microglia and phagocytic macrophages. Thus, trauma-induced central nervous system (CNS) inflammation, regardless of strain, occurs rapidly at the site of injury and involves the activation of resident and recruited immune cells. In regions rostral or caudal to the epicenter, prolonged activation of inflammatory cells occurs preferentially in white matter and primarily consists of activated microglia and astrocytes. Differences were observed in the magnitude and duration of macrophage activation between Sprague-Dawley (SD) and Lewis (LEW) rats throughout the lesion. Increased expression of complement type 3 receptors (OX42) and macrophage-activation antigens (ED1) persisted for longer times in LEW rats while expression of MHC class II molecules was attenuated in LEW compared to SD rats at all times examined. Variations in the onset and duration of T-lymphocyte infiltration also were observed between strains with twice as many T-cells present in the lesion epicenter of Lewis rats by 3 days post-injury. These strain-specific findings potentially represent differences in corticosteroid regulation of immunity and may help predict a range of functional neurologic consequences affected by neuroimmune interactions.

Differential expression of MHC class II antigen in the contused rat spinal cord.

Following contusion injury to the dorsal surface of thoracic rat spinal cord, major histocompatibility complex (MHC) class II (Ia) antigen expression by microglia was evaluated throughout the developing lesion. Past investigations of various central nervous system (CNS) lesions have examined short-term or acute sequelae of post-traumatic Ia expression. This report demonstrates that in animals allowed to recover for 18 (sub-chronic) and 45 (chronic) days post-injury, MHC class II antigen is expressed differently at rostral and caudal extents of the lesion as compared with the lesion's epicenter. Following contusion injury to the thoracic spinal cord, sub-chronically injured animals demonstrated Ia-positive microglial staining throughout the white matter rostral and caudal to the epicenter of the lesion, whereas Ia-positive microglia and/or perivascular cells are localized within the gray matter adjacent to it. MHC class II immunoreactivity is down-regulated on microglia at chronic survival times but clusters of Ia-positive macrophages are prominent in regions of maximal degeneration at the epicenter of the lesion. Our findings support the theory that two distinct populations of macrophages participate in resolving traumatic injury. One population is the parenchymal CNS microglia and the other is presumably exudate macrophages derived from the blood. Furthermore, the immunocompetence of these cells as measured by MHC expression may be differentially regulated. This hypothesis is based on differences in Ia-positive staining observed between microglia and macrophages over time concomitant with differences in the spatial distribution of these cell types.

Alterations in immune cell phenotype and function after experimental spinal cord injury.

Traumatic injury to the spinal cord initiates a cascade of inflammatory-mediated injury and repair processes within the nervous system. In parallel, spinal injury could influence peripheral mechanisms of host defense (e.g., wound healing, antibody production) by altering lymphocyte phenotype and function. The goal of this study was to evaluate the physiological impact of spinal contusion injury on phenotypic and functional indices of lymphocyte activation. A flow cytometric time-course analysis of lymphocytes isolated from lymph node and spleen revealed an increase in CD4+ and a decrease in CD8+ lymphocytes during the first week post injury. The functional potential of lymphocytes was also evaluated based on their ability to proliferate in the presence of a biologically relevant antigen (myelin basic protein, MBP) or a lymphocyte mitogen. The data revealed increased proliferation to MBP by 3 days postinjury in lymphocytes isolated from lymph node but not spleen. By 1 week postinjury, increased proliferation to mitogen was noted in both the lymph node and the spleen suggesting a general increase in lymphocyte reactivity during this time interval. Circulating corticosterone (CORT), an endogenous glucocorticoid with significant effects on lymphocyte phenotype and function, was elevated within 24 h after spinal cord injury (SCI) and remained above control levels throughout the duration of our studies (up to 1 month postinjury). The present data suggest injury-associated changes in immune cell phenotype and function paralleled by the activation of the hypothalamic-pituitary-adrenal (HPA) axis.

Analysis of TGF-beta 1 gene expression in contused rat spinal cord using quantitative RT-PCR.

We have used northern blot analysis and quantitative reverse transcription polymerase chain reaction (RT-PCR) to determine the postinjury expression profile of the transforming growth factor-beta 1 (TGF-beta 1) gene in the contused rat spinal cord. Spectrophotometric estimates of total sample RNA and quantitative analyses of cyclophilin mRNA using RT-PCR served as controls for comparisons between samples. No changes in cyclophilin gene expression were found at any postinjury survival times. The results of the TGF-beta 1 analyses, which were carried out on spinal cord samples taken at postinjury intervals ranging from 6 h to 10 days, show that the amount of TGF-beta 1 mRNA present in spinal cord increases rapidly following injury, reaching maximum levels 7 days postinjury. Unoperated control samples contained approximately 2 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA. By 1 day postinjury, the amount of TGF-beta 1 mRNA in the cord had increased by a factor of 2.5 to 5 x 10(8) molecules/0.5 microgram total RNA. At 7 days postinjury, there were approximately 15 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA. By 10 days postinjury the amount of TGF-beta 1 mRNA present in the spinal cord had declined to 8 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA, a value similar to that observed at 3 days postinjury. The roles that TGF-beta 1 might play in modifying cellular responses in injured spinal cord are discussed.

Traumatic spinal cord injury produced by controlled contusion in mouse.

Previous work from this laboratory has described a rat spinal cord injury (SCI) model in which the mid-thoracic spinal cord is subjected to a single rapid and calibrated displacement at the site of a dorsal laminectomy. Injury is initiated at the tip of a vertical shaft driven by an electromagnetic shaker. Transducers arranged in series with the shaft record the patterns of displacement and force during the impact sequence. In the present study, this device and the relevant surgical procedures were adapted to produce a spinal contusion injury model in laboratory mice. The signal generator for the injury device has also been converted to a computer-controlled interface to permit extension of the model to other laboratories. Mice were subjected to SCI across a range of severities by varying the amplitude of displacement and the magnitude of measured preload force on the dural surface. A moderate injury produced by displacement of 0.5 mm over 25 msec resulted in initial paralysis and recovery of locomotion with chronic deficits in hindlimb function. The magnitude of the peak force, impulse, power, and energy generated at impact were correlated with behavioral outcome at 1 day postinjury, while peak displacement and impulse were the best predictors of behavioral outcome at 28 days postinjury. The shape of the force recording proved to be a highly sensitive measure of subtle variations in the spinal compartment that were otherwise difficult to detect in this small species. The results demonstrate that the electromagnetic spinal cord injury device (ESCID) can be used to produce a well-controlled contusion injury in mice. The unique features of controlled displacement and monitoring of the biomechanical parameters at the time of impact provide advantages of this model for reducing outcome variability. Use of this model in mice with naturally occurring and genetically engineered mutations will facilitate understanding of the molecular mechanisms of pathophysiology following traumatic spinal cord injury.

Elevated serum titers of proinflammatory cytokines and CNS autoantibodies in patients with chronic spinal cord injury.

This study characterized the proinflammatory cytokines, interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFalpha), the antiinflammatory cytokines, IL-4 and IL-10, autoantibodies specific for GM1 ganglioside (anti-GM1), IgG and IgM, and myelin-associated glycoprotein (anti-MAG), in the sera of infection-free, chronic (>12 months), traumatically injured SCI patients (n = 24). Healthy able-bodied subjects (n = 26) served as controls. The proinflammatory cytokines and anti-GM1 antibodies were of particular interest as they have been implicated in an autoimmune "channelopathy" component to central and peripheral conduction deficits in various chronic neuroinflammatory diseases. Antibody and cytokine titers were established using enzyme-linked immunosorbent assays (ELISA). The mean anti-GM(1) (IgM) titer value for the SCI group was significantly higher (p < 0.05) than controls. The SCI group also demonstrated significantly higher titers (p < 0.05) of IL-2 and TNF alpha than controls. No differences were found between the SCI group and control group mean levels of IL-4 or IL-10. Overall, the serum of 57% of SCI patients contained increased levels of autoantibodies or proinflammatory cytokines relative to control values. These results provide preliminary support for the hypothesis that chronic immunological activation in the periphery occurs in a subpopulation of chronic SCI patients. It remains to be established whether elevated serum titers of proinflammatory cytokines and autoantibodies against GM1 are beneficial to the patients or whether they are surrogate markers of a channelopathy that compounds the neurological impairment associated with traumatic axonopathy or myelinopathy.

Elevation of the neurotoxin quinolinic acid occurs following spinal cord trauma.

Excitatory amino acid neurotoxicity and the inflammatory response are suspected as mediators of some of the pathological sequelae occurring as a result of spinal cord injury. Here we report temporal and regional increases of the NMDA receptor agonist, quinolinic acid (QUIN), in an experimental model of spinal contusion injury. These changes occurred at a time when the blood-brain barrier is known to be dysfunctional and the activation state and density of microglia and macrophages are increased. Thus, alterations in tissue QUIN levels may occur as a result of secondary activation of CNS inflammatory cells or from peripherally derived sources across a damaged blood-brain barrier.

Let's talk: patterns and correlates of social support among temporary employees.

The authors explored how temporary employees exchanged communications with supervisor, peers, and family and friends regarding positively job-related, negatively job-related, and non-job-related contents. The authors also examined roles of communication in coping with insecure job experiences. Survey results from 112 temporary employees working in various organizations provided evidence that communication contents were differentially related to work anxiety and life satisfaction for temporary employees. It was found that work anxiety increased when employees engaged in communication pertaining to negative job-related contents. Furthermore, the positive relationship between life satisfaction and positive communication with coworkers was observed only for the temporary employees who also had a permanent job. Implications for staffing temporary employees and suggestions for studying communication effects are discussed.

Criteria used to judge obese persons in the workplace.

Researchers have speculated that employers are less likely to hire obese persons for more publicly visible jobs, although this hypothesis remains untested. In the present study, 54 undergraduate students rated 40 jobs on several items, including the likelihood they would hire an obese person for each job. Multidimensional scaling showed a one-dimensional solution, labeled as physical activity, with participants less likely to hire obese persons for more active jobs. For hiring likelihood ratings for jobs at either end of the dimension appear to be most similar for men and individuals with more positive attitudes toward obese persons versus women and individuals with more negative attitudes toward obese persons. Implications for both theory and practice are discussed.