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Age-related changes in TuJ1 and NF 200 expression in human ears. A: TuJ1 + neurons in a paraffin section from the middle turn of a young adult mouse. B-C: TuJ1 + neurons in the middle turns from (B) a 38-year-old donor (H18) and (C) a 69-year-old donor (H4). A marked decrease in TuJ1 immunostaining (asterisks) was seen in SGNs of the older ear. The white arrow indicates a neuron with a relatively normal level of TuJ1 immunoreactivity. Nuclei were counterstained with PI (red). D, E, F: A marked decrease of NF200 immunoreactivity was seen in many SGNs of older donors. NF200 + neurons (green) from the middle turns of (D) a 46-year-old (H26), (E) a 67-year-old (H19), and (F) a 91-year-old donor (H3). Asterisks indicate SGNs with a lower level of NF200 immunoreactivity. White arrows indicate SGNs with a level of NF200 immunoreactivity more similar to that of SGNs from the 46-year-old donor shown in D. Note the punctuate inclusions (white arrowheads) in the cytoplasm of some NF200 + SGNs indicating that neurofilament aggregation occurs in older ears. G, H, I: Confocal images showing a reduction in both the number of NF200 + neurons and decreased immunostaining intensity for NF200 in most surviving SGNs in older donors. Asterisks indicate SGNs with a lower level of NF200 immunoreactivity. A white arrow points to an SGN with a normal level of NF200 immunoreactivity. Punctuate inclusions (white arrowheads) were seen in the cytoplasm of many NF200 + SGNs in the 91-year-old donor. Scale bars, 8 mm in C (applies to A-C); 7 mm in I (applies to D-I). doi:10.1371/journal.pone.0034500.g008
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Age-related hearing loss (presbyacusis) is the most common type of hearing impairment. One of the most consistent pathological changes seen in presbyacusis is the loss of spiral ganglion neurons (SGNs). Defining the cellular and molecular basis of SGN degeneration in the human inner ear is critical to gaining a better understanding of the pathophys...
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... sections of both middle-aged and older human temporal bones. A marked decrease in immunostaining intensity for TuJ1 was seen in SGNs of the older group compared to the middle-aged group (Figs. 8B, C). Immunereactivity for another neuronal structural protein, neurofilament 200 (NF200), also was markedly diminished in the aged human spiral ganglion (Figs. 8D-I). In addition, NF200 + punctuate inclusions were seen in the cytoplasm of many SGNs in the older group, particularly within the basal turns (Figs. 8E, F, ...
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... compared to the middle-aged group (Figs. 8B, C). Immunereactivity for another neuronal structural protein, neurofilament 200 (NF200), also was markedly diminished in the aged human spiral ganglion (Figs. 8D-I). In addition, NF200 + punctuate inclusions were seen in the cytoplasm of many SGNs in the older group, particularly within the basal turns (Figs. 8E, F, ...
Citations
... Generally, these results confirmed that DMF/RSF/P:P conduits could inhibit SC pyroptosis on the cell surface. Subsequently, we examined the effects of culture supernatants from the five groups of Schwann cells on PC12 cell function by analyzing two neuronal structural proteins, [48] neurofilament 200 (NF200) and the III tubulin isotype (Tuj1). The NF200 and Tuj1 levels in the RSF/DMF and RSF/DMF/ES groups were greater than those in the RSF/DMSO and RSF/ES groups, indicating that the DMF/RSF/P:P conduits with ES indirectly promoted PC12 cell function by inhibiting Schwann cell pyroptosis in vitro ( Figure 3F,G). ...
Peripheral nerve injury (PNI) remains a challenging area in regenerative medicine. Nerve guide conduit (NGC) transplantation is a common treatment for PNI, but the prognosis of NGC treatment is unsatisfactory due to 1) neuromechanical unmatching and 2) the intra‐conduit inflammatory microenvironment (IME) resulting from Schwann cell pyroptosis and inflammatory‐polarized macrophages. A neuromechanically matched NGC composed of regenerated silk fibroin (RSF) loaded with poly(3,4‐ethylenedioxythiophene): poly(styrene sulfonate) (P:P) and dimethyl fumarate (DMF) are designed, which exhibits a matched elastic modulus (25.1 ± 3.5 MPa) for the peripheral nerve and the highest 80% elongation at break, better than most protein‐based conduits. Moreover, the NGC can gradually regulate the intra‐conduit IME by releasing DMF and monitoring sciatic nerve movements via piezoresistive sensing. The combination of NGC and electrical stimulation modulates the IME to support PNI regeneration by synergistically inhibiting Schwann cell pyroptosis and reducing inflammatory factor release, shifting macrophage polarization from the inflammatory M1 phenotype to the tissue regenerative M2 phenotype and resulting in functional recovery of neurons. In a rat sciatic nerve crush model, NGC promoted remyelination and functional and structural regeneration. Generally, the DMF/RSF/P:P conduit provides a new potential therapeutic approach to promote nerve repair in future clinical treatments.
... Bilirubin also causes demyelination other than directly leads to neuron apoptosis [45]. Myelin deficits have been associated with hearing loss in animal models as well as in the auditory nerve in aging human temporal bones [46]. ...
... The relative differentiation from oligodendrocytes, which MBP indicates, is comparable to the inferior colliculus [15]. An age-related decline in MBP is known from studies of the auditory nerve in mice [48]. It should be noted that NSCs are not a homogeneous cell population but a combination of diverse subpopulations. ...
Neural stem cells (NSCs) have been recently identified in the neonatal rat medial geniculate body (MGB). NSCs are characterized by three cardinal features: mitotic self-renewal, formation of progenitors, and differentiation into all neuroectodermal cell lineages. NSCs and the molecular factors affecting them are particularly interesting, as they present a potential target for treating neurologically based hearing disorders. It is unclear whether an NSC niche exists in the rat MGB up to the adult stage and which neurogenic factors are essential during maturation. The rat MGB was examined on postnatal days 8, 12, and 16, and at the adult stadium. The cardinal features of NSCs were detected in MGB cells of all age groups examined by neurosphere, passage, and differentiation assays. In addition, real-time quantitative polymerase chain reaction arrays were used to compare the mRNA levels of 84 genes relevant to NSCs and neurogenesis. In summary, cells of the MGB display the cardinal features of NSCs up to the adult stage with a decreasing NSC potential over time. Neurogenic factors with high importance for MGB neurogenesis were identified on the mRNA level. These findings should contribute to a better understanding of MGB neurogenesis and its regenerative capacity.
... On the other hand, some degenerative myelin changes including myelin splitting and myelin balloons were also observed in the aging mouse MNTB (data not shown). Indeed, numerous previous studies have shown various age-related myelin alterations in mice, rats, gerbils, and monkeys, as well as geriatric people, such as local splitting of the myelin sheath along the major dense line to accommodate dense cytoplasm, formation of myelin ballooning, partial loss of myelin and demyelination (Attia et al., 2019;Bouhrara et al., 2021;Faddis and McGinn, 1997;Feldman and Peters, 1998;Li et al., 2009;Li et al., 2017;Peters, 2002;Peters et al., 2000;Peters et al., 2001;Sugiyama et al., 2002;Xing et al., 2012). These observations indicated age-related, degenerative myelin degradation during the aging process of the brain. ...
... The balloons range from 10-20 µm in diameter(Feldman and Peters, 1998). Similar degeneration is reported in old rats and mice(Sugiyama et al., 2002;Xing et al., 2012). Thus, our observations in the mice SNpc match those of the classical studies. ...
Opposing phenomena of both oligodendrogliosis and oligodendroglial degeneration is reported in the striatum of MPTP-injected mice model of Parkinson’s disease (PD); reducing the clarity on their contribution to PD pathology. Furthermore, the role of oligodendroglia of substantia nigra pars compacta (SNpc), in pathogenesis and differential susceptibility to PD, is not known. In our earlier study, the MPTP-sensitive young C57BL/6J showed loss of 50-60% of SNpc neurons following MPTP, whereas the MPTP-resistant CD-1 showed a loss of 15-17%, suggesting better neuroprotection in the latter. We now investigate the effects of age and MPTP on oligodendroglial and myelin sheath ultrastructure, in SNpc, by electron microscopy as well as myelin-associated protein CNPase by semi-quantitative confocal microscopy and immunoblotting.
MPTP-induced mitochondrial shrinkage suggests toxicity to mitochondria and dilation of Golgi-complex saccules indicates protein packaging deficits, in the oligodendroglia of young C57BL/6J. Contrariwise in CD-1, presence of elongated mitochondria and multiple polyribosomes may suggest preserved ultrastructure. The organelles were affected at middle age in C57BL/6J but at old age in CD-1. The neuropil of SNpc in both strains harbored unmyelinated and lightly myelinated fibers. The enhanced density of myelin fibers following MPTP, suggests re-myelination of surviving neurons. MPTP aggravated myelin decompaction at middle age in C57BL/6J, but at old age in CD-1. Upregulation of CNPase in MPTP-injected middle-aged CD-1 suggests attempt at compensation. Thus, oligodendroglia of the MPTP-sensitive strain shows degenerative features, while those of MPTP-resistant strain show compensatory capabilities against neuroinflammatory milieu; till middle age. It is pertinent to reconsider oligodendroglial involvement in PD.
... The net effect of strial dysfunction is the same as that of synaptopathy: fewer action potentials driving the auditory pathway (Lang et al., 2010;Schmiedt et al., 2002). Therefore, reductions of the endocochlear potential (Gratton et al., 1997;Schulte & Schmiedt, 1992) (or auditory nerve demyelination; Xing et al., 2012) may also cause reductions of wave-I amplitude and slope. Although strial degeneration is unlikely to occur for older people with normal audiograms (Wu et al., 2020), it remains possible that the age-related decline in ABR wave-I amplitude or slope reflects strial degeneration (and/or demyelination) instead of or in addition to synaptopathy or deafferentation. ...
Older people often show auditory temporal processing deficits and speech-in-noise intelligibility difficulties even when their audiogram is clinically normal. The causes of such problems remain unclear. Some studies have suggested that for people with normal audiograms, age-related hearing impairments may be due to a cognitive decline, while others have suggested that they may be caused by cochlear synaptopathy. Here, we explore an alternative hypothesis, namely that age-related hearing deficits are associated with decreased inhibition. For human adults (N = 30) selected to cover a reasonably wide age range (25–59 years), with normal audiograms and normal cognitive function, we measured speech reception thresholds in noise (SRTNs) for disyllabic words, gap detection thresholds (GDTs), and frequency modulation detection thresholds (FMDTs). We also measured the rate of growth (slope) of auditory brainstem response wave-I amplitude with increasing level as an indirect indicator of cochlear synaptopathy, and the interference inhibition score in the Stroop color and word test (SCWT) as a proxy for inhibition. As expected, performance in the auditory tasks worsened (SRTNs, GDTs, and FMDTs increased), and wave-I slope and SCWT inhibition scores decreased with ageing. Importantly, SRTNs, GDTs, and FMDTs were not related to wave-I slope but worsened with decreasing SCWT inhibition. Furthermore, after partialling out the effect of SCWT inhibition, age was no longer related to SRTNs or GDTs and became less strongly related to FMDTs. Altogether, results suggest that for people with normal audiograms, age-related deficits in auditory temporal processing and speech-in-noise intelligibility are mediated by decreased inhibition rather than cochlear synaptopathy.
... In a study, MBP detected early myelination in the rat brain stem [29], and MBP detected myelination in the second trimester human fetal spinal cord [30]. MBP is a well-known marker to detect demyelination in multiple sclerosis, human auditory nerve, and some other neurodegenerative diseases [31][32][33][34]. We found severe alterations in p-SNI rats, including neuroinflammation followed by monocyte-derived macrophage activation, as revealed by the CD68 marker. ...
Demyelination is a common sign of peripheral nerve injuries (PNIs) caused by damage to the myelin sheath surrounding axons in the sciatic nerve. There are not many methods to induce demyelination in the peripheral nervous system (PNS) using animal models. This study describes a surgical approach using a single partial sciatic nerve suture to induce demyelination in young male Sprague Dawley (SD) rats. After the post-sciatic nerve injury (p-SNI) to the sciatic nerve, histology and immunostaining show demyelination or myelin loss in early to severe phases with no self-recovery. The rotarod test confirms the loss of motor function in nerve-damaged rats. Transmission electron microscopic (TEM) imaging of nerve-damaged rats reveals axonal atrophy and inter-axonal gaps. Further, administration of Teriflunomide (TF) to p-SNI rats resulted in the restoration of motor function, repair of axonal atrophies with inter-axonal spaces, and myelin secretion or remyelination. Taken together, our findings demonstrate a surgical procedure that can induce demyelination in the rat sciatic nerve, which is then remyelinated after TF treatment.
... Interestingly, we also observed that TuJ1, a neuronal marker, showed an aggregation-like pattern with condensed TuJ1 signal restricted to neuron cell bodies unilaterally in the Tmprss3 A306T/A306T mice, which may further support previous findings showing cellular and molecular basis of SGN degeneration and the subsequent decline of the auditory nerve function in presbyacusis. 74 While the physiological outcomes of TuJ1 aggregation is unknown, both the altered TuJ1 localization and the neuronal loss strongly support the direct impact of the TMPRSS3 mutation A306T on the SGN and the rescue effect by AAV2-hTMPRSS3 local delivery. Mutations in TMPRSS3 cause DFNB8 with HL that is postlingual and progressive, and DFNB10 with congenital profound HL. ...
Patients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10. For these patients, cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knockin mouse model with a frequent human DFNB8 TMPRSS3 mutation. The Tmprss3A306T/A306T homozygous mice display delayed onset progressive hearing loss similar to human DFNB8 patients. Using AAV2 as a vector to carry a human TMPRSS3 gene, AAV2-hTMPRSS3 injection in the adult knockin mouse inner ear results in TMPRSS3 expression in the hair cells and the spiral ganglion neurons. A single AAV2-hTMPRSS3 injection in Tmprss3A306T/A306T mice of an average age of 18.5 months leads to sustained rescue of the auditory function to a level similar to wild-type mice. AAV2-hTMPRSS3 delivery rescues the hair cells and the spiral ganglions neurons. This study demonstrates successful gene therapy in an aged mouse model of human genetic deafness. It lays the foundation to develop AAV2-hTMPRSS3 gene therapy to treat DFNB8 patients, as a standalone therapy or in combination with cochlear implantation.
... Interestingly, we also observed that TuJ1, a neuronal marker, showed an aggregationlike pattern with condensed TuJ1 signal restricted to neuron cell bodies unilaterally in the Tmprss3 A306T/A306T mice, which may further support previous findings showing cellular and molecular basis of SGN degeneration and the subsequent decline of the auditory nerve function in presbyacusis. 75 While the physiological outcomes of TuJ1 aggregation is unknown, both the altered TuJ1 localization and the neuronal loss strongly support that direct impact of the TMPRSS3 mutation A306T on the spiral ganglion neurons and the rescue effect by AAV2-hTMPRSS3 local delivery. Mutations in TMPRSS3 cause DFNB8 with hearing loss that is postlingual and progressive, and DFNB10 with congenital profound hearing loss. ...
... doi: bioRxiv preprint labyrinths were removed, and cochleae were immediately soaked in ice-cold 4% PFA in PBS. Cochleae were fixed for 2 hrs at RT on a shaker and washed for 3 x 10 min in 0.1% Tween-20 in PBS (PBT20), and subsequently dehydrated in a graded MeOH series (50,75, and 100% in PBT20, 10 min for each grade). At the same time, probes were pre-warmed to 40°C for 10 min to allow aggregates to dissolve and then cooled down to RT. Protease III solution and Amplifiers I-IV were allowed to equilibrate to RT. ...
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Patients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10 for whom cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knock-in mouse model with a frequent human DFNB8 TMPRSS3 mutation. The Tmprss3 A306T/A306T homozygous mice display delayed onset progressive hearing loss similar to human DFNB8 patients. Using AAV2 as a vector to carry a human TMPRSS3 gene, AAV2-h TMPRSS3 injection in the adult knock-in mouse inner ears results in TMPRSS3 expression in the hair cells and the spiral ganglion neurons. A single AAV2-h TMPRSS3 injection in aged Tmprss3 A306T/A306T mice leads to sustained rescue of the auditory function, to a level similar to the wildtype mice. AAV2-h TMPRSS3 delivery rescues the hair cells and the spiral ganglions. This is the first study to demonstrate successful gene therapy in an aged mouse model of human genetic deafness. This study lays the foundation to develop AAV2-h TMPRSS3 gene therapy to treat DFNB8 patients, as a standalone therapy or in combination with cochlear implantation.
... In this study, we decided to stain against MBP because it specifically labels MBP, which is crucial for myelin development and compaction, and it is one of the most abundant structural proteins in myelin (Park et al., 2016). MBP immunoreactivity is used for the visualization of myelinated fibers in humans and other mammals (Ahn et al., 2017;Park et al., 2016;Tanaka et al., 1995;Xing et al., 2012), and it has been used to validate results from MRI techniques (Chang et al., 2016;Soustelle et al., 2019). Other traditional techniques used to measure myelin in zebra finches such as Gallyas silver stain (Herrmann & Bishof, 1986;Kafitz et al., 1992;Karten et al., 2013) or the modified Gallyas myelin stain (Champoux et al., 2021) also provide high resolution visualization, but the procedure requires familiarity in the use of pyridine which introduces a potential hazard. ...
The role of myelination in the development of motor control is widely known, but its role in the development of cognitive abilities is less understood. Here we examined sex differences in the development of myelination of structures and tracts that support song learning and production in songbirds. We collected brains from 63 young male and female zebra finches (Taeniopygia guttata) over four stages of development that correspond to different stages of song learning. Using a myelination marker (myelin basic protein), we measured the development of myelination in three different nuclei of the vocal control system (HVC, RA, LMAN) and 2 tracts (HVC‐RA and LMV). We found that the myelination of the vocal control nuclei and tracts is sex related and male biased. In males, the patterns of myelination were age‐dependent, asynchronous in rate and progression and associated with the development of song learning and production. In females, myelination of vocal control nuclei was low or absent and did not significantly change with age. Sex differences in myelination of the HVC‐RA tract were large and emerged late in development well after sex differences in the size of vocal control brain regions are established. Myelination of this tract in males coincides with the age of song crystallization. Overall, the changes in myelination in the vocal control areas and tracts measured are region‐, age‐, and sex‐specific and are consistent with sex differences in song development. This article is protected by copyright. All rights reserved
