Trigeminal Sensory Supply Is Essential for Motor Recovery after Facial Nerve Injury.

Recovery of mimic function after facial nerve transection is poor. The successful regrowth of regenerating motor nerve fibers to reinnervate their targets is compromised by (i) poor axonal navigation and excessive collateral branching, (ii) abnormal exchange of nerve impulses between adjacent regrowing axons, namely axonal crosstalk, and (iii) insufficient synaptic input to the axotomized facial motoneurons. As a result, axotomized motoneurons become hyperexcitable but unable to discharge. We review our findings, which have addressed the poor return of mimic function after facial nerve injuries, by testing the hypothesized detrimental component, and we propose that intensifying the trigeminal sensory input to axotomized and electrophysiologically silent facial motoneurons improves the specificity of the reinnervation of appropriate targets. We compared behavioral, functional, and morphological parameters after single reconstructive surgery of the facial nerve (or its buccal branch) with those obtained after identical facial nerve surgery, but combined with direct or indirect stimulation of the ipsilateral infraorbital nerve. We found that both methods of trigeminal sensory stimulation, i.e., stimulation of the vibrissal hairs and manual stimulation of the whisker pad, were beneficial for the outcome through improvement of the quality of target reinnervation and recovery of vibrissal motor performance.

Motor, sensitive, and vegetative recovery in rats with compressive spinal-cord injury after combined treatment with erythropoietin and whole-body vibration.

BACKGROUND: Physical therapy with whole body vibration (WBV) following compressive spinal cord injury (SCI) in rats restores density of perisomatic synapses, improves body weight support and leads to a better bladder function. The purpose of the study was to determine whether the combined treatment with WBV plus erythropoietin (EPO) would further improve motor, sensory and vegetative functions after SCI in rats. METHODS: Severe compressive SCI at low thoracic level was followed by a single i.p. injection of 2,5µg (250 IU) human recombinant EPO. Physical therapy with WBV started on 14th day after injury and continued over a 12-week post injury period. Locomotor recovery, sensitivity tests and urinary bladder scores were analysed at 1, 3, 6, 9, and 12 weeks after SCI. The closing morphological measurements included lesion volume and numbers of axons in the preserved perilesional neural tissue bridges (PNTB). RESULTS: Assessment of motor performance sensitivity and bladder function revealed no significant effects of EPO when compared to the control treatments. EPO treatment neither reduced the lesion volume, nor increased the number of axons in PNTB. CONCLUSIONS: The combination of WBV + EPO exerts no positive effects on hind limbs motor performance and bladder function after compressive SCI in rats.

Neutralizing BDNF and FGF2 injection into denervated skeletal muscle improve recovery after nerve repair.

BACKGROUND: After facial nerve injury and surgical repair in rats, recovery of vibrissal whisking is associated with a high proportion of mono-innervated neuro-muscular junctions (NMJs). Our earlier work with Sprague Dawley (SD)/Royal College of Surgeons (RCS) rats, which are blind and spontaneously restore NMJ-monoinnervation and whisking, showed correlations between functional recovery and increase of fibroblast growth factor-2 (FGF2) and brain-derived neurotrophic factor (BDNF) in denervated vibrissal muscles. METHODS: We used normally sighted rats (Wistar), in which NMJ-polyinnervation is highly correlated with poor whisking recovery, and injected the vibrissal muscle levator labii superioris (LLS) with combinations of BDNF, anti-BDNF, and FGF2 at different postoperative periods after facial nerve injury. RESULTS: Rats receiving anti-BDNF+FGF2 showed low NMJ-polyinnervation and best recovery of whisking amplitude. CONCLUSIONS: Restoration of target reinnervation after peripheral nerve injury requires a complex mixture of trophic factors with a specific time course of availability for each of them.

Whole body vibration (WBV) following spinal cord injury (SCI) in rats: Timing of intervention.

BACKGROUND: Following spinal cord injury (SCI), exercise training provides a wide range of benefits and promotes activity-dependent synaptic plasticity. Whole body vibration (WBV) in SCI patients improves walking and spasticity as well as bone and muscle mass. However, little is known about the effects of timing or frequency of intervention. OBJECTIVE: To determine which WBV-onset improves locomotor and bladder functions and influences synaptic plasticity beneficially. METHODS: SCI was followed by WBV starting 1, 7, 14, 28 days after injury (WBV1, WBV7, etc.) and continued for 12 weeks. Intact animals and those receiving SCI but no WBV (No WBV), SCI plus WBV twice daily (2×WBV) and SCI followed by passive hindlimb flexion-extension (PFE) served as controls. Locomotor [BBB rating, foot stepping angle (FSA) and rump-height index (RHI)] as well as bladder function were determined at 1, 3, 6, 9, and 12 weeks. Following perfusion fixation at 12 weeks, lesion volume and immunofluorescence for astrogliosis (GFAP), microglia (IBA1) and synaptic vesicles (synaptophysin, SYN) were determined. RESULTS: Compared to the No WBV group, the WB7 and WBV14 groups showed significantly faster speeds of BBB score recovery though this effect was temporary. Considering RHI we detected a sustained improvement in the WBV14 and PFE groups. Bladder function was better in the WBV14, WBV28, 2×WBV and PFE groups. Synaptophysin levels improved in response to WBV7 and WBV14, but worsened after WBV28 in parallel to an increased IBA1 expression. Correlation- and principal components analysis revealed complex relationships between behavioural (BBB, FSA, RHI) and morphological (GFAP, IBA1, SYN) measurements. CONCLUSIONS: WBV started 14 days after SCI provides the most benefit (RHI, bladder); starting at 1day after SCI provides no benefit and starting at 28 days may be detrimental. Increasing the intensity of WBV to twice daily did not provide additional benefit.

Growth of Murine Splenic Tissue Is Suppressed by Lymphotoxin ß-Receptor Signaling (LTßR) Originating from Splenic and Non-Splenic Tissues.

Development and maintenance of secondary lymphoid organs such as lymph nodes and spleen essentially depend on lymphotoxin ß-receptor (LTßR) signaling. It is unclear, however, by which molecular mechanism their size is limited. Here, we investigate whether the LTßR pathway is also growth suppressing. By using splenic tissue transplantation it is possible to analyze a potential contribution of LTßR signaling inside and outside of the implanted tissue. We show that LTßR signaling within the endogenous spleen and within non-splenic tissues both significantly suppressed the regeneration of implanted splenic tissue. The suppressive activity positively correlated with the total number of LTßR expressing cells in the animal (regenerate weights of 115 ± 8 mg in LTßR deficient recipients and of 12 ± 9 mg in wild-type recipients), affected also developed splenic tissue, and was induced but not executed via LTßR signaling. Two-dimensional differential gel electrophoresis and subsequent mass spectrometry of stromal splenic tissue was applied to screen for potential factors mediating the LTßR dependent suppressive activity. Thus, LTßR dependent growth suppression is involved in regulating the size of secondary lymphoid organs, and might be therapeutically used to eradicate tertiary lymphoid tissues during autoimmune diseases.

Comparison of trophic factors' expression between paralyzed and recovering muscles after facial nerve injury. A quantitative analysis in time course.

After peripheral nerve injury, recovery of motor performance negatively correlates with the poly-innervation of neuromuscular junctions (NMJ) due to excessive sprouting of the terminal Schwann cells. Denervated muscles produce short-range diffusible sprouting stimuli, of which some are neurotrophic factors. Based on recent data that vibrissal whisking is restored perfectly during facial nerve regeneration in blind rats from the Sprague Dawley (SD)/RCS strain, we compared the expression of brain derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), insulin growth factors 1 and 2 (IGF1, IGF2) and nerve growth factor (NGF) between SD/RCS and SD-rats with normal vision but poor recovery of whisking function after facial nerve injury. To establish which trophic factors might be responsible for proper NMJ-reinnervation, the transected facial nerve was surgically repaired (facial-facial anastomosis, FFA) for subsequent analysis of mRNA and proteins expressed in the levator labii superioris muscle. A complicated time course of expression included (1) a late rise in BDNF protein that followed earlier elevated gene expression, (2) an early increase in FGF2 and IGF2 protein after 2 days with sustained gene expression, (3) reduced IGF1 protein at 28 days coincident with decline of raised mRNA levels to baseline, and (4) reduced NGF protein between 2 and 14 days with maintained gene expression found in blind rats but not the rats with normal vision. These findings suggest that recovery of motor function after peripheral nerve injury is due, at least in part, to a complex regulation of lesion-associated neurotrophic factors and cytokines in denervated muscles. The increase of FGF-2 protein and concomittant decrease of NGF (with no significant changes in BDNF or IGF levels) during the first week following FFA in SD/RCS blind rats possibly prevents the distal branching of regenerating axons resulting in reduced poly-innervation of motor endplates.

Cytokinesis failure and successful multipolar mitoses drive aneuploidy in glioblastoma cells.

Glioblastoma (GB) is the most frequent human brain tumor and is associated with a poor prognosis. Multipolar mitosis and spindles have occasionally been observed in cultured glioblastoma cells and in glioblastoma tissues, but their mode of origin and relevance have remained unclear. In the present study, we investigated a novel GB cell line (SGB4) exhibiting mitotic aberrations and established a functional link between cytokinesis failure, centrosome amplification, multipolar mitosis and aneuploidy in glioblastoma. Long-term live cell imaging showed that >3% of mitotic SGB4 cells underwent multipolar mitosis (tripolar>tetrapolar>pentapolar). A significant amount of daugther cells generated by multipolar mitosis were viable and completed several rounds of mitosis. Pedigree analysis of mitotic events revealed that in many cases a bipolar mitosis with failed cytokinesis occurred prior to a multipolar mitosis. Additionally, we observed that SGB4 cells were also able to undergo a bipolar mitosis after failed cytokinesis. Colchicine-induced mitotic arrest and metaphase spreads demonstrated that SGB4 cells had a modal chromosome number of 58 ranging from 23 to 170. Approximately 82% of SGB4 cells were hyperdiploid (47-57 chromosomes) or hypotriploid (58-68 chromosomes). In conclusion, SGB4 cells passed through multipolar cell divisions and generated viable progeny by reductive mitoses. Our results identified cytokinesis failure occurring before and after multipolar or bipolar mitoses as important mechanisms to generate chromosomal heterogeneity in glioblastoma cells.

Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy.

Cyclase-associated proteins are highly conserved proteins that have a role in the regulation of actin dynamics. Higher eukaryotes have two isoforms, CAP1 and CAP2. To study the in vivo function of CAP2, we generated mice in which the CAP2 gene was inactivated by a gene-trap approach. Mutant mice showed a decrease in body weight and had a decreased survival rate. Further, they developed a severe cardiac defect marked by dilated cardiomyopathy (DCM) associated with drastic reduction in basal heart rate and prolongations in atrial and ventricular conduction times. Moreover, CAP2-deficient myofibrils exhibited reduced cooperativity of calcium-regulated force development. At the microscopic level, we observed disarrayed sarcomeres with development of fibrosis. We analyzed CAP2's role in actin assembly and found that it sequesters G-actin and efficiently fragments filaments. This activity resides completely in its WASP homology domain. Thus CAP2 is an essential component of the myocardial sarcomere and is essential for physiological functioning of the cardiac system, and a deficiency leads to DCM and various cardiac defects.

Differential glycomics of epithelial membrane glycoproteins from urinary exovesicles reveals shifts toward complex-type N-glycosylation in classical galactosemia.

A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause profound activity loss of the enzyme and acute toxic effects mediated by accumulating metabolic intermediates of galactose in newborns induced by dietary galactose. However, even on a severely galactose-restricted diet, patients develop serious long-term complications of the CNS and ovaries, which may result from damaging perturbations in cell biology caused by endogenously synthezised galactose. Under galactose stress, the cosubstrate of GALT, galactose-1-phosphate, accumulates and disturbs catabolic and anabolic pathways of the carbohydrate metabolism with potential effects on protein glycosylation and membrane localization of glycoprotein receptors, like the epidermal growth factor receptor. To address this issue in view of a cellular pathomechanism, we performed a differential semiquantitative N-glycomics study of membrane proteins. A suitable noninvasive cellular material derived from epithelial plasma membranes was found in urinary exovesicles and in the shed Tamm-Horsfall protein. By applying matrix-assisted laser ionization mass spectrometry on permethylated, PNGaseF released N-glycans, we demonstrate that GALT deficiency is associated with dramatic shifts from prevalent high-mannose-type glycans found in healthy subjects toward complex-type N-linked glycosylation in patients. These N-glycosylation shifts were observed on exosomal N-glycoproteins but not on the Tamm-Horsfall glycoprotein, which showed predominant high-mannose-type glycosylation with M6.

TNF receptor-1 is required for the formation of splenic compartments during adult, but not embryonic life.

Lymphotoxin ß-receptor (LTßR) and TNF receptor-1 (TNFR1) are important for the development of secondary lymphoid organs during embryonic life. The significance of LTßR and TNFR1 for the formation of lymphoid tissue during adult life is not well understood. Immunohistochemistry, morphometry, flow cytometry, and laser microdissection were used to compare wild-type, LTßR(-/-), TNFR1(-/-) spleens with splenic tissue that has been newly formed 8 wk after avascular implantation into adult mice. During ontogeny, LTßR is sufficient to induce formation of the marginal zone, similar-sized T and B cell zones, and a mixed T/B cell zone that completely surrounded the T cell zone. Strikingly, in adult mice, the formation of splenic compartments required both LTßR and TNFR1 expression, demonstrating that the molecular requirements for lymphoid tissue formation are different during embryonic and adult life. Thus, interfering with the TNFR1 pathway offers the possibility to selectively block the formation of ectopic lymphoid tissue and at the same time to spare secondary lymphoid organs such as spleen and lymph nodes. This opens a new perspective for the treatment of autoimmune and inflammatory diseases.

Dendritic cells release HLA-B-associated transcript-3 positive exosomes to regulate natural killer function.

NKp30, a natural cytotoxicity receptor expressed on NK cells is critically involved in direct cytotoxicity against various tumor cells and directs both maturation and selective killing of dendritic cells. Recently the intracellular protein BAT3, which is involved in DNA damage induced apoptosis, was identified as a ligand for NKp30. However, the mechanisms underlying the exposure of the intracellular ligand BAT3 to surface NKp30 and its role in NK-DC cross talk remained elusive. Electron microscopy and flow cytometry demonstrate that exosomes released from 293T cells and iDCs express BAT3 on the surface and are recognized by NKp30-Ig. Overexpression and depletion of BAT3 in 293T cells directly correlates with the exosomal expression level and the activation of NK cell-mediated cytokine release. Furthermore, the NKp30-mediated NK/DC cross talk resulting either in iDC killing or maturation was BAT3-dependent. Taken together this puts forward a new model for the activation of NK cells through intracellular signals that are released via exosomes from accessory cells. The manipulation of the exosomal regulation may offer a novel strategy to induce tumor immunity or inhibit autoimmune diseases caused by NK cell-activation.

Number and function of circulating human antigen presenting cells regulated by sleep.

STUDY OBJECTIVES: There is evidence that sleep facilitates the adaptive immune response to infectious agents and, thereby, supports immunologic memory. The effect might be attained by sleep-induced changes in the number and function of dendritic cells (DCs), which play a key role in the initiation of the immune response. This study aimed to dissociate effects of sleep and circadian rhythm on circulating numbers of DC precursors, ie, CD14+CD16- and CD14(dim)CD16+ monocytes, myeloid dendritic cell precursors (pre-mDC), and plasmacytoid dendritic cells (PDC) and on 2 key cytokines produced by these cells, ie, interleukin (IL)-12 and interferon (IFN)-alpha. DESIGN: In a within-subject cross-over design, human subjects were examined on 2 occasions, ie, during a normal sleep-wake cycle and during 24 hours of wakefulness. Blood was sampled every 1.5 hours during nighttime and every 3 hours during daytime. SETTING: Experiments took place under controlled laboratory conditions. PARTICIPANTS: Twenty-seven healthy men aged between 18 and 30 years. MEASUREMENTS AND RESULTS: Compared with wakefulness, sleep was associated with a striking increase in the number of pre-mDC producing IL-12, which is a main inducer of Th1 responses. In addition, sleep slightly decreased PDC and also T cell counts but did not affect IFN-alpha production by PDC. Sleep, however, substantially decreased numbers of CD14(dim)CD16+ monocytes, probably reflecting increased margination of the cells upon a sleep-related drop in catecholamine release. CONCLUSIONS: Our data identify pre-mDC producing IL-12 as a basic target of sleep that is most closely related to mature APC function and whereby sleep can effectively enhance adaptive immune responses.

Metallophilic macrophages are lacking in the thymus of lymphotoxin-beta receptor-deficient mice.

Lymphotoxin-beta receptor (LTbetaR) axis plays a crucial role in development and compartmentalization of peripheral lymphatic organs. But, it is also required for the appropriate function and maintenance of structural integrity of the thymus: in LTbetaR-deficient animals the clonal deletion of autoreactive lymphocytes is impaired and differentiation of thymic medullary epithelial cells is disturbed. In this study, using several markers, we showed that thymic metallophilic macrophages were lacking in LTbetaR-deficient mice. In tumor necrosis factor receptor-I (p55)-deficient mice (which we used as positive control) thymic metallophilic cells were located, similarly as in normal mice, in the thymic cortico-medullary zone at the junction of cortex and medulla. These findings show that LTbetaR is necessary for maintenance of metallophilic macrophages in the thymus and provide further evidence that these cells may represent a factor involved in thymic negative selection.

T cells are required for the peripheral phase of B-cell maturation.

B-lymphocyte maturation is considered to be independent of the thymus. However, there is circumstantial evidence suggesting that it may be impaired in nude animals that lack the thymus. Our study shows that the proportion of immature B-lymphocyte subsets (CD90(high) IgM(high) and CD90(high) IgM(low)) was significantly increased, whereas that of mature B-lymphocyte subsets (CD90- IgM(low) and CD90- IgM(high)) was decreased in the blood and lymph nodes of nude rats. In addition, the expression of major histocompatibility complex class II, intercellular adhesion molecule-1, CD44 and l-selectin was significantly down-regulated both on immature and mature B-lymphocyte subsets. After implantation of thymic tissue under the kidney capsule of nude rats the block in B-lymphocyte maturation was alleviated and the expression of surface molecules was normalized. Comparable effects were seen after the adoptive transfer of T lymphocytes. Thus, we show that in nude rats B cells do not mature properly because of the lack of T-cell help and that T lymphocytes are required for the peripheral phase of B-lymphocyte maturation, as well as for the appropriate expression of surface molecules. This should be considered when treating patients with T-cell deficiencies.

Blood lymphocytes, monocytes and NK cells modulate their expression of CD44, ICAM-1, LFA-1 and MHC class II after arrival into lymphoid organs.

Adhesion molecules expressed on surface membranes of lymphocytes and other leukocytes enable their entry into the lymphoid and other tissues. However, little is known about molecules that govern the transit of leukocytes through the parenchyma of lymphoid organs proper. We show that in comparison to blood leukocytes, the corresponding cells isolated from lymphoid organs, i.e., lymph nodes and spleen, have a significantly augmented expression of certain surface molecules. The helper and cytotoxic subsets of T cells, as well as B cells, display the increased expression of CD44, ICAM-1 and LFA-1, whereas B cells additionally show the augmented expression of MHC class II. In comparison with blood monocytes, splenic monocytes show the increased expression of ICAM-1 and MHC class II molecules. When compared with blood NK cells, splenic NK cells only show the increased expression of ICAM-1. The molecules, which we show to be up regulated upon the entry of leukocytes into lymphoid organs, could be involved in their retention within the tissue via cell-cell or cell-extracellular matrix interactions and in control of their transit through lymphoid tissues.

Analyzing the migration of labeled T cells in vivo: an essential approach with challenging features.

T cells are involved in the pathogenesis of many diseases. To exert a pathological effect, T cells enter the tissues. We show that the determination of their entry site requires isolation of the respective T cell population, injection into genetically un-manipulated animals, and identification of the cells in vivo at various time points after injection. We indicate variables influencing in vivo migration experiments artificially, and outline how resulting problems can be either avoided or taken into account. Reviewing experiments performed according to the outlined criteria reveals two types of migration patterns for T cell subsets in vivo: 1). Naïve and memory T cells enter lymphoid and non-lymphoid organs in comparable numbers, but selectively accumulate in lymphoid tissues over time, 2). Effector T cells, too, enter lymphoid and non-lymphoid organs in comparable numbers. However, most of them die within 24 hours. Depending on the presence of cytokines, chemokines and extracellular matrix compounds they are able to survive, thereby preferentially accumulating in their target tissues. This information might help to understand the role of migration in the pathogenesis of T cell mediated diseases.