Hypoxia-Mediated Soluble Fms-Like Tyrosine Kinase 1 Increase Is Not Attenuated in Interleukin 6-Deficient Mice.

The soluble fms-like tyrosine kinase 1 (sFlt-1), known to be increased in the serum of preeclamptic patients, is a relevant factor in causing maternal symptoms like hypertension and proteinuria. In this study, we aimed to reveal whether hypoxia is a cause of increased sFlt-1 levels and inflammation markers in vivo and whether these symptoms can be attenuated by interleukin 6 (IL-6) depletion. For this purpose, pregnant wild-type (wt) mice or IL-6(-/-) mice on embryonic day 16 were placed under either normoxic (20.9% oxygen) or hypoxic (6% oxygen) conditions for 6 hours. This led to a rise of sFlt-1 levels in maternal serum, independent of the IL-6 status of the dam. Increased maternal sFlt-1 serum levels were, however, not due to an increase in sFlt-1 messenger RNA levels in the placenta. Moreover, there was no increase in inflammatory markers in neither wt mice nor IL-6(-/-) mice. This suggests that hypoxia alone does not contribute to the induction of an inflammatory placenta. Also, the hypoxia-induced rise in sFlt-1 levels seems not to be mediated by IL-6 in vivo.

CNN3 regulates trophoblast invasion and is upregulated by hypoxia in BeWo cells.

CNN3 is an ubiquitously expressed F-actin binding protein, shown to regulate trophoblast fusion and hence seems to play a role in the placentation process. In this study we demonstrate that CNN3 levels are upregulated under low oxygen conditions in the trophoblast cell line BeWo. Since hypoxia is discussed to be a pro-migratory stimulus for placental cells, we examined if CNN3 is involved in trophoblast invasion. Indeed, when performing a matrigel invasion assay we were able to show that CNN3 promotes BeWo cell invasion. Moreover, CNN3 activates the MAPKs ERK1/2 and p38 in trophoblast cells and interestingly, both kinases are involved in BeWo invasion. However, when we repeated the experiments under hypoxic conditions, CNN3 did neither promote cell invasion nor MAPK activation. These results indicate that CNN3 promotes invasive processes by the stimulation of ERK1/2 and/or p38 under normoxic conditions in BeWo cells, but seems to have different functions at low oxygen levels. We further speculated that CNN3 expression might be altered in human placentas derived from pregnancies complicated by IUGR and preeclampsia, since these placental disorders have been described to go along with impaired trophoblast invasion. Our studies show that, at least in our set of placenta samples, CNN3 expression is neither deregulated in IUGR nor in preeclampsia. In summary, we identified CNN3 as a new pro-invasive protein in trophoblast cells that is induced under low oxygen conditions.

Manual stimulation of the whisker pad after hypoglossal-facial anastomosis (HFA) using a Y-tube conduit does not improve recovery of whisking function.

Facial nerve injury is a common clinical trauma involving long-term functional deficits with facial asymmetry leading to associated psychological issues and social hardship. We have recently shown that repair by hypoglossal-facial or facial-facial nerve surgical end-to-end anastomosis and suture [hypoglossal-facial anastomosis (HFA) or facial-facial anastomosis (FFA)] results in collateral axonal branching, polyinnervation of neuromuscular junctions (NMJs) and poor function. We have also shown that another HFA repair procedure using an isogenic Y-tube (HFA + Y-tube) and involving a 10-mm gap reduces collateral axonal branching, but fails to reduce polyinnervation. Furthermore, we have previously demonstrated that manual stimulation (MS) of facial muscles after FFA or HFA reduces polyinnervation of NMJs and improves functional recovery. Here, we examined whether HFA + Y-tube and MS of the vibrissal muscles reduce polyinnervation and restore function. Isogenic Y-tubes were created using abdominal aortas. The proximal hypoglossal nerve was inserted into the long arm and sutured to its wall. The distal zygomatic and buccal facial nerve branches were inserted into the two short arms and likewise sutured to their walls. Manual stimulation involved gentle stroking of the vibrissal muscles by hand mimicking normal whisker movement. We evaluated vibrissal motor performance using video-based motion analysis, degree of collateral axonal branching using double retrograde labeling and the quality of NMJ reinnervation in target musculature using immunohistochemistry. MS after HFA + Y-tube reduced neither collateral branching, nor NMJ polyinnervation. Accordingly, it did not improve recovery of function. We conclude that application of MS after hypoglossal-facial nerve repair using an isogenic Y-tube is contraindicated: it does not lead to functional recovery but, rather, worsens it.

Whole-body vibration improves functional recovery in spinal cord injured rats.

Whole-body vibration (WBV) is a relatively novel form of exercise used to improve neuromuscular performance in healthy individuals. Its usefulness as a therapy for patients with neurological disorders, in particular spinal cord injury (SCI), has received little attention in clinical settings and, surprisingly, even less in animal SCI models. We performed severe compression SCI at a low-thoracic level in Wistar rats followed by daily WBV starting 7 (10 rats) or 14 (10 rats) days after injury (WBV7 and WBV14, respectively) and continued over a 12-week post-injury period. Rats with SCI but no WBV training (sham, 10 rats) and intact animals (10 rats) served as controls. Compared to sham-treated rats, WBV did not improve BBB score, plantar stepping, or ladder stepping during the 12-week period. Accordingly, WBV did not significantly alter plantar H-reflex, lesion volume, serotonergic input to the lumbar spinal cord, nor cholinergic or glutamatergic inputs to lumbar motoneurons at 12 weeks after SCI. However, compared to sham, WBV14, but not WBV7, significantly improved body weight support (rump-height index) during overground locomotion and overall recovery between 6-12 weeks and also restored the density of synaptic terminals in the lumbar spinal cord at 12 weeks. Most remarkably, WBV14 led to a significant improvement of bladder function at 6-12 weeks after injury. These findings provide the first evidence for functional benefits of WBV in an animal SCI model and warrant further preclinical investigations to determine mechanisms underpinning this noninvasive, inexpensive, and easily delivered potential rehabilitation therapy for SCI.

Use of a Y-tube conduit after facial nerve injury reduces collateral axonal branching at the lesion site but neither reduces polyinnervation of motor endplates nor improves functional recovery.

BACKGROUND: Despite increased understanding of peripheral nerve regeneration, functional recovery after surgical repair remains disappointing. A major contributing factor is the extensive collateral branching at the lesion site, which leads to inaccurate axonal navigation and aberrant reinnervation of targets. OBJECTIVE: To determine whether the Y tube reconstruction improved axonal regrowth and whether this was associated with improved function. METHODS: We used a Y-tube conduit with the aim of improving navigation of regenerating axons after facial nerve transection in rats. RESULTS: Retrograde labeling from the zygomatic and buccal branches showed a halving in the number of double-labeled facial motor neurons (15% vs 8%; P < .05) after Y tube reconstruction compared with facial-facial anastomosis coaptation. However, in both surgical groups, the proportion of polyinnervated motor endplates was similar (≈ 30%; P > .05), and video-based motion analysis of whisking revealed similarly poor function. CONCLUSION: Although Y-tube reconstruction decreases axonal branching at the lesion site and improves axonal navigation compared with facial-facial anastomosis coaptation, it fails to promote monoinnervation of motor endplates and confers no functional benefit.

Hypoglossal-facial anastomosis (HFA) over a 10 mm gap bridged by a Y-tube-conduit enhances neurite regrowth and reduces collateral axonal branching at the lesion site.

PURPOSE: The outcome of severe peripheral nerve injuries requiring surgical repair (transection and suture) is usually poor. Recent work suggests that direct suture of nerves increases collagen production and provides unfavourable conditions for a proper axonal regrowth. We tested whether entubulation of the hypoglossal nerve into a Y-tube conduit connecting it with the zygomatic and buccal facial nerve branches would improve axonal pathfinding at the lesion site, quality of muscle reinnervation and recovery of vibrissal whisking. METHODS: For hypoglossal-facial anastomosis (HFA) over a Y-tube (HFA-Y-tube) the proximal stump of the hypoglossal nerve was entubulated and sutured into the long arm of a Y-tube (isogeneic abdominal aorta with its bifurcation). The zygomatic and buccal facial branches were entubulated and sutured to the short arms of the Y-tube. Restoration of vibrissal motor performance, degree of collateral axonal branching at the lesion site and quality of neuro-muscular junction (NMJ) reinnervation were compared to animals receiving HFA-Coaptation (no entubulation) after 4 months. RESULTS: HFA-Y-tube reduced collateral axonal branching. However it failed to reduce the proportion of polyinnervated NMJ and did not improve functional outcome when compared to HFA-Coaptation. CONCLUSION: Elimination of compression by tightly opposed nerve fragments improved axonal pathfinding. However, biometric analysis of vibrissae movements did not show positive effects suggesting that polyneuronal reinnervation - rather than collateral branching - may be the critical limiting factor. Since polyinnervation of muscle fibers is activity-dependent and can be manipulated, the present findings raise hopes that clinically feasible and effective therapies after HFA could be soon designed and tested.

Objective measures of motor dysfunction after compression spinal cord injury in adult rats: correlations with locomotor rating scores.

Precise assessment of motor deficits after traumatic spinal cord injury (SCI) in rodents is crucial for understanding the mechanisms of functional recovery and testing therapeutic approaches. Here we analyzed the applicability to a rat SCI model of an objective approach, the single-frame motion analysis, created and used for functional analysis in mice. Adult female Wistar rats were subjected to graded compression of the spinal cord. Recovery of locomotion was analyzed using video recordings of beam walking and inclined ladder climbing. Three out of four parameters used in mice appeared suitable: the foot-stepping angle (FSA) and the rump-height index (RHI), measured during beam walking, and for estimating paw placement and body weight support, respectively, and the number of correct ladder steps (CLS), assessing skilled limb movements. These parameters, similar to the Basso, Beattie, and Bresnahan (BBB) locomotor rating scores, correlated with lesion volume and showed significant differences between moderately and severely injured rats at 1-9 weeks after SCI. The beam parameters, but not CLS, correlated well with the BBB scores within ranges of poor and good locomotor abilities. FSA co-varied with RHI only in the severely impaired rats, while RHI and CLS were barely correlated. Our findings suggest that the numerical parameters estimate, as intended by design, predominantly different aspects of locomotion. The use of these objective measures combined with BBB rating provides a time- and cost-efficient opportunity for versatile and reliable functional evaluations in both severely and moderately impaired rats, combining clinical assessment with precise numerical measures.