The human middle ear in motion: 3D visualization and quantification using dynamic synchrotron-based X-ray imaging.

The characterization of the vibrations of the middle ear ossicles during sound transmission is a focal point in clinical research. However, the small size of the structures, their micrometer-scale movement, and the deep-seated position of the middle ear within the temporal bone make these types of measurements extremely challenging. In this work, dynamic synchrotron-based X-ray phase-contrast microtomography is used on acoustically stimulated intact human ears, allowing for the three-dimensional visualization of entire human eardrums and ossicular chains in motion. A post-gating algorithm is used to temporally resolve the fast micromotions at 128 Hz, coupled with a high-throughput pipeline to process the large tomographic datasets. Seven ex-vivo fresh-frozen human temporal bones in healthy conditions are studied, and the rigid body motions of the ossicles are quantitatively delineated. Clinically relevant regions of the ossicular chain are tracked in 3D, and the amplitudes of their displacement are computed for two acoustic stimuli.

Setting thresholds is not enough: Beach litter as indicator of poor environmental status in the southern Adriatic Sea.

This study deals with the issue of beach litter pollution in the context of the Descriptor 10 of the Marine Strategy Framework Directive Good Environmental Status of EU waters and Ecological objective 10, Common indicator 22 of IMAP. Analyses of the amount, distribution and categorization of beach litter were conducted on nine beaches during 108 surveys covering the area of 206.620 m2 in Albania, Italy and Montenegro. Our findings showed that the level of beach litter pollution on south Adriatic beaches is significantly above the adopted threshold values, with a median item numbers of 327, 258 and 234 per 100 m of beach stretch for Albania, Italy and Montenegro, respectively. It can be concluded that, when it comes to beach litter pollution, GES has not been achieved. Given the defined baseline and threshold values at the EU level, the process of reducing the total amount of marine litter in southern Adriatic Sea will be very challenging and needs urgent and specific actions.

Ca2+-binding protein 2 inhibits Ca2+-channel inactivation in mouse inner hair cells.

Ca2+-binding protein 2 (CaBP2) inhibits the inactivation of heterologously expressed voltage-gated Ca2+ channels of type 1.3 (CaV1.3) and is defective in human autosomal-recessive deafness 93 (DFNB93). Here, we report a newly identified mutation in CABP2 that causes a moderate hearing impairment likely via nonsense-mediated decay of CABP2-mRNA. To study the mechanism of hearing impairment resulting from CABP2 loss of function, we disrupted Cabp2 in mice (Cabp2LacZ/LacZ ). CaBP2 was expressed by cochlear hair cells, preferentially in inner hair cells (IHCs), and was lacking from the postsynaptic spiral ganglion neurons (SGNs). Cabp2LacZ/LacZ mice displayed intact cochlear amplification but impaired auditory brainstem responses. Patch-clamp recordings from Cabp2LacZ/LacZ IHCs revealed enhanced Ca2+-channel inactivation. The voltage dependence of activation and the number of Ca2+ channels appeared normal in Cabp2LacZ/LacZ mice, as were ribbon synapse counts. Recordings from single SGNs showed reduced spontaneous and sound-evoked firing rates. We propose that CaBP2 inhibits CaV1.3 Ca2+-channel inactivation, and thus sustains the availability of CaV1.3 Ca2+ channels for synaptic sound encoding. Therefore, we conclude that human deafness DFNB93 is an auditory synaptopathy.

Skin derived precursor Schwann cells improve behavioral recovery for acute and delayed nerve repair.

Previous work has shown that infusion of skin-derived precursors pre-differentiated into Schwann cells (SKP-SCs) can remyelinate injured and regenerating axons, and improve indices of axonal regeneration and electrophysiological parameters in rodents. We hypothesized that SKP-SC therapy would improve behavioral outcomes following nerve injury repair and tested this in a pre-clinical trial in 90 rats. A model of sciatic nerve injury and acellular graft repair was used to compare injected SKP-SCs to nerve-derived Schwann cells or media, and each was compared to the gold standard nerve isograft repair. In a second experiment, rats underwent right tibial nerve transection and received either acute or delayed direct nerve repair, with injections of either 1) SKP-SCs distal to the repair site, 2) carrier medium alone, or 3) dead SKP-SCs, and were followed for 4, 8 or 17weeks. For delayed repairs, both transected nerve ends were capped and repaired 11weeks later, along with injections of cells or media as above, and followed for 9 additional weeks (total of 20weeks). Rats were serially tested for skilled locomotion and a slip ratio was calculated for the horizontal ladder-rung and tapered beam tasks. Immediately after nerve injury and with chronic denervation, slip ratios were dramatically elevated. In the GRAFT repair study, the SKP-SC treated rats showed statistically significant improvement in ladder rung as compared to all other groups, and exhibited the greatest similarity to the sham controls on the tapered beam by study termination. In the ACUTE repair arm, the SKP-SC group showed marked improvement in ladder rung slip ratio as early as 5weeks after surgery, which was sustained for the duration of the experiment. Groups that received media and dead SKP-SCs improved with significantly slower progression. In the DELAYED repair arm, the SKP-SC group became significantly better than other groups 7weeks after the repair, while the media and the dead SKP-SCs showed no significant improvement in slip ratios. On histomorphometrical analysis, SKP-SC group showed significantly increased mean axon counts while the percent myelin debris was significantly lower at both 4 and 8weeks, suggesting that a less inhibitory micro-environment may have contributed to accelerated axonal regeneration. For delayed repair, mean axon counts were significantly higher in the SKP-SC group. Compound action potential amplitudes and muscle weights were also improved by cell therapy. In conclusion, SKP-SC therapy improves behavioral recovery after acute, chronic and nerve graft repair beyond the current standard of microsurgical nerve repair.

The impact of motor axon misdirection and attrition on behavioral deficit following experimental nerve injuries.

Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r(2)=.67), motor neuron counts (attrition) (p<.0001, r(2)=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize functional recovery. It also demonstrates that clinically relevant neuroma-in-continuity injuries can now also be subjected to experimental investigation.

The cytoskeletal adapter protein 4.1G organizes the internodes in peripheral myelinated nerves.

Myelinating Schwann cells regulate the localization of ion channels on the surface of the axons they ensheath. This function depends on adhesion complexes that are positioned at specific membrane domains along the myelin unit. Here we show that the precise localization of internodal proteins depends on the expression of the cytoskeletal adapter protein 4.1G in Schwann cells. Deletion of 4.1G in mice resulted in aberrant distribution of both glial adhesion molecules and axonal proteins that were present along the internodes. In wild-type nerves, juxtaparanodal proteins (i.e., Kv1 channels, Caspr2, and TAG-1) were concentrated throughout the internodes in a double strand that flanked paranodal junction components (i.e., Caspr, contactin, and NF155), and apposes the inner mesaxon of the myelin sheath. In contrast, in 4.1G(-/-) mice, these proteins "piled up" at the juxtaparanodal region or aggregated along the internodes. These findings suggest that protein 4.1G contributes to the organization of the internodal axolemma by targeting and/or maintaining glial transmembrane proteins along the axoglial interface.

The function of glutamatergic synapses is not perturbed by severe knockdown of 4.1N and 4.1G expression.

AMPA-type glutamate receptors mediate fast excitatory synaptic transmission in the vertebrate brain. Their surface expression at synapses between neurons is regulated in an activity-dependent and activity-independent manner. The protein machinery that regulates synaptic targeting, anchoring and turnover of AMPA receptors consists of several types of specialized scaffolding proteins. The FERM domain scaffolding proteins 4.1G and 4.1N were previously suggested to act jointly in binding and regulating synaptic trafficking of the AMPA receptor subunits GluR1 and GluR4. To determine the functions of 4.1G and 4.1N in vivo, we generated a mutant mouse line that lacks 4.1G entirely and expresses 4.1N at 22% of wild-type levels. These mice had combined 4.1G and 4.1N protein expression in the hippocampus at 12% of wild-type levels (equivalent to 8-10% of combined GluR1 and GluR4 expression levels). They show a moderate reduction in synaptosomal expression levels of the AMPA receptor subunit GluR1 at 3 weeks of age, but no change in basic glutamatergic synaptic transmission and long-term potentiation in the hippocampus. Our study indicates that 4.1G and 4.1N do not have a crucial role in glutamatergic synaptic transmission and the induction and maintenance of long-term plastic changes in synaptic efficacy.