Figure 1.
Decline of auditory nerve function in aged CBA/CaJ mice.
A: Comparisons of ABR thresholds for wave I obtained from 1–3 month-old and 23–27 month-old mice. It showed significant ABR threshold shifts in older mice across all frequencies tested (5.6, 8, 11.3, 16, 22.6, 32, and 40 kHz). No ABR response was detected at 40 kHz in older mice. B, C, D: Mean wave I amplitude vs. level functions. The data show increased ABR thresholds and decreased wave I amplitudes in aged mice at 11.3, 16, and 22.6 kHz. All data are presented as mean ± SEM. Asterisks indicated statistically significant differences at the indicated frequencies (p<0.05).
Figure 2.
Age-related ultrastructural changes in CBA/CaJ mouse spiral ganglion.
A, B: Normal type I (I) SGNs and myelinating Schwann cells (S) in the middle turn of a young adult mouse. B shows a higher magnification view of the boxed area in A. Chromatin is evenly distributed in the prominent nucleus and the neuronal perikaryon is surrounded by a myelin sheath and by the peripheral process of a Schwann cell. The majority of myelin sheaths enclosing type I SGNs are tightly laminated with no discontinuities. Although a separation of myelin sheath layers enveloping SGNs was occasionally seen in young adult mice (arrow), no discontinuities in the myelin sheath were seen in the young mice. C: A section from the basal turn of a 27-month-old mouse showing SGNs in various stage of degeneration. A degenerating SGN surrounded by a thinned and broken myelin sheath (black arrows) and numerous electron dense inclusions are present in the cytoplasm. On the left, a gap (white arrow) is present in the myelin sheath enclosing a relatively healthy appearing SGN. On the right, debris from a dead SGN is enclosed by a myelin sheath and a Schwann cell process (asterisk). D, E, F: Abnormalities of the myelin sheath in the middle turn of another 27-month-old mouse. Black arrows indicate areas of broken (D, F) and loose (E) myelin lamellae enclosing both degenerative and normal SGNs. The dense inclusions in the cytoplasm of Schwann cells are indicated by white arrows (E). G, H, I: Degenerative changes in the middle turn of a 27-month-old mouse. Abnormal partially collapsed or folded myelin sheaths (black arrows) and balloon-like features (white arrowhead) are seen surrounding the perikarya of SGNs (G, H) and their processes (I). J: Numerous electron dense inclusions in the cytoplasm of a Schwann cell in the center (white arrows). The Schwann cell associated SGN is surrounded by a broken myelin sheath (black arrow). K, L, M, N: Abnormal collapsed and folded myelin sheaths (white arrow), broken myelin sheaths (black arrow), balloon-like features (white arrowhead), and vacuole-like spaces (black arrowheads) surrounding the perikarya of SGNs (J,L) and auditory nerve processes within RC (L) and OSL (M,N). L is a higher magnification view of the boxed area in K. A white arrow in M points electron dense inclusions in the cytoplasm of a Schwann cell. All images in J–N were taken from a 28-month-old mouse. Scale bars = 2 µm in A–N.
Figure 3.
Immunohistochemical localization of myelin basic protein (MBP) in young adult CBA/CaJ mouse ears.
A, B, C: Confocal images of SGNs labeled with anti-class III β-tubulin (TuJ1, red) and anti-MBP (green) within the medial portion of Rosenthal's canal (RC) in the apical (A,B) and basal (C) turns of a one-month-old mouse. TuJ1 is a neuronal marker that preferentially labels the cytoplasm of type I SGNs and their processes. MBP labeling of myelin sheaths revealed a honeycomb-like staining pattern in the spiral ganglion. D, E, F: Myelinated peripheral processes in the osseous spiral lamina (OSL) of the basal turn of a three-month-old mouse stained strongly for MBP. A white arrow points to the habenula perforata (Hb) where auditory fibers lose their myelinated sheaths. G, H, I: MBP+ fibers in the lateral portion of RC in the basal turn of a three-month-old mouse. MBP+ myelinated fibers and MBP− unmyelinated fibers form intraganglionic spiral bundles. J,K,L: MBP+ fibers in the glial transition zone (GTZ) of the modiolus (MO) ensheathed by oligodendrocytes (CNS) and Schwann cells (PNS). Nuclei were counterstained with PI (red). Organ of Corti, OCT; Scala tympani, ST. Scale bars, 12 µm in L (applies to A–L).
Figure 4.
Age-related changes in MBP expression in the CBA/CaJ mouse spiral ganglion.
A: MBP+ myelin sheaths (green) enclose type I SGNs in Rosenthal's canal of the basal turn of a one month-old mouse. Intact MBP+ myelin sheaths (asterisks) are seen enveloping most neurons. Propidium iodide (PI) nuclear counterstaining (red) reveals nuclear profiles of both SGNs and Schwann cells. SGNs are identifiable by their larger, more weakly stained spherical nuclei whereas the nuclei of Schwann cells are recognized by their irregular shape and more intense staining. B, C: Abnormalities in MBP expression patterns in Rosenthal's canal of aged mice. Numerous SGNs are enclosed only partially by a MBP+ myelin sheath (arrows) and some SGNs appear to lose their MBP+ myelin sheath. Asterisks indicate two SGNs with normal-looking MBP+ myelin sheaths. D, E: Confocal images of myelin sheaths enveloping neurons from young (D) and aged (E) mice. Intact MBP+ myelin sheaths are indicated by asterisks while broken MBP+ myelin sheaths are indicated by white arrows. F: Quantitative analysis of SGNs with intact MBP+ myelin sheaths in young and aged mouse ears. The MBP+ myelin sheath was considered intact if it enclosed more than 80% of the outline of the perikarya. A significant decline in the percentage of intact MBP+ myelin sheath was found in all three cochlear turns in the aged mouse ears compared to young controls (n = 5–7 per group; P<0.01; asterisk). Scale bar, 10 µm in C (applies to A–C); 10 µm in E (applies to D–E).
Figure 5.
Age-related degeneration of MBP+ nerve fibers in aged CBA/CaJ mice.
A, B: Confocal images of the MBP+ nerve fibers in the osseous spiral lamina of a one-month-old (A) and a 26-month-old (B) mouse. White lines indicate the areas where MBP+ fibers were counted (please see Materials and Methods for detailed information on MBP+ fiber counting). C: Mean density of MBP+ fibers within osseous spiral lamina of the apical, middle, and basal turns from young and aged mouse ears (n = 3–5 per group). A significant age-related reduction of MBP+ fiber density was found within the apical and basal turn (P<0.05; asterisk). D, E, F: Degeneration of MBP+ myelin in the auditory nerve of a 26-month-old mouse. Dual labeling with anti-MBP (green) and anti-class III β-tubulin (TuJ1, red) indicates that partial demyelination (white arrowheads) occurs in the nerve fibers within the OSL. Scale bar, 8 µm in F (applies to A–F).
Figure 6.
Immunolocalization of MBP in middle-aged human ears.
Left panel: Low-power view of apical and middle turns from a 46-year-old donor illustrating auditory nerve fibers in different regions stained positively for MBP (green). Nuclei were counterstained with PI (red). Right panel: Enlarged images corresponding to the boxed areas labeled A–F in the left panel. A: MBP+ fibers located within Rosenthal's canal (RC) of the apical turn. B: MBP+ fibers in the central projection (CP) of the auditory nerve within the modiolus close to the apical turn. C: MBP+ fibers in the peripheral process (PP) of the auditory nerve within the osseous spiral lamina (OSL). D: MBP+ intraganglionic spiral bundle (ISB) fibers in the middle turn. E: MBP+ fibers located within Rosenthal's canal of the middle turn. F: Transverse section of MBP+ fibers in the CP within the modiolus close to the middle turn. Additional abbreviations: OCT, organ of Corti; SL, spiral ligament; SM, scala media; ST, scala tympani; SV, scala vestibuli. Scale bar, 7 µm in F applies to A–F.
Table 1.
Temporal bone donor information and immunoreactivity for MBP antibody.
Figure 7.
Age-related changes in MBP expression in human ears.
A–B: A thin MBP+ myelin layer (arrows) around the perikarya of some type I neurons in both the middle (A) and basal (B) turns of ears from middle-aged donors. The inset in A shows a type I neuron with an intact MBP+ myelin layer at higher magnification. C: A MBP+ myelin layer was not observed enveloping any neurons in a mid-modiolar section taken from a 74-year-old donor. There was also a marked loss of MBP+ fibers between neurons and a reduction in the number of PI+ glial cell nuclei, relative to the middle-aged specimens. D, E: Confocal images of the MBP+ myelin layer (arrows) around the perikarya of some type I neurons in the spiral ganglion from a 38-year-old and a 46-year-old donor. F: A MBP+ myelin layer was not observed in a mid-modiolar section taken from a 91-year-old donor. Scale bar, 12 µm in C (applies to A–C); 12 µm in F (applies to D–F).
Figure 8.
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 µm in C (applies to A–C); 7 µm in I (applies to D–I).
Figure 9.
Age-related reduction of MBP+ fibers and immunostaining for MBP in human ears.
A–L; N–O: A significant loss of MBP+ fibers occurred with age in both the peripheral and central portions of the auditory nerve. MBP+ nerve fibers are illustrated in peripheral (A, B) and central (C, D) portions of the auditory nerve in the ears taken from middle-aged (A–D) and old (E–H) donors. White boxes with associated letters in M illustrated the regions of the cochlea depicted in A–H. Counts of MBP+ fibers in both peripheral (I, J) and central (K, L) projections revealed a statistically significant reduction in fiber density in older compared to middle aged human cochleas using an unpaired t-test (*p<0.05). N, O: A reduction in the intensity of MBP immunoreactivity was seen in many myelinated fibers of a 91-year-old donor (white arrowheads). Scala vestibuli, SV; Scala tympani, ST. Scale bar, 10 µm in E (applies to A, E); 12 µm in H (applies to B–D; F–H); 12 µm in O (applies to N, O).
Table 2.
Antibody Characterization.