Dysfunction of the inner ear is the most common cause of sensorineural hearing loss, which is the most common sensory deficit worldwide. Conventional imaging modalities are unable to depict the microanatomy of the human inner ear, hence the need to explore novel imaging modalities. We provide the first characterization of the polarization dependent optical properties of human cochlear sections using quantitative polarized light microscopy (qPLM). Eight pediatric cadaveric cochlear sections, aged 0 (term) to 24 months, were selected from the US National Temporal Bone Registry, imaged with qPLM and analyzed using Image J. Retardance of the bony otic capsule and basilar membrane were substantially higher than that of the stria vascularis, spiral ganglion neurons, organ of Corti and spiral ligament across the half turns of the spiraling cochlea. qPLM provides quantitative information about the human inner ear, and awaits future exploration in vivo.
© 2015 Optical Society of America
Hearing loss affects over 36 million Americans  and 600 million people worldwide . Although the majority of sensorineural hearing loss originates from the inner ear, individual cells and structure within the human inner ear cannot yet be imaged using current state-of-the-art clinical tools, including computed tomography (CT) and magnetic resonance imaging (MRI) scans. Today, the only source of information about the cellular basis of human deafness is cadaveric human temporal bones that house the inner ear. To enable future cellular-level intracochlear imaging in alive humans, we have been exploring optical imaging tools [3,4] because optics provides higher spatial resolution at a lower cost than techniques based on ionizing radiation or magnetic resonance.
We have recently demonstrated that quantitative polarized light microscopy (qPLM) detects differences in polarization dependent optical properties of key intracochlear structures, as evaluated using unstained mouse cochlear sections , while being faster and cheaper than immunohistochemistry. The current study explores the utility of qPLM in imaging human cochlear sections. Polarized light microscopy (PLM) is based on the principle that biological specimens alter the polarization state of the polarized light that passes through them. When the refractive index of the specimen varies anisotropically with the polarization state and propagation direction of an incoming light wave (a property known as birefringence), nonhomogeneous alterations in wave propagation velocity lead to a phase shift on orthogonal polarization states of the transmitted light wave. This phase shift is referred to as sample retardance and it can be measured in nanometers, r, or as a phase angle δ, where δ = 2πr/λ and λ is the wavelength of the light. Specimens that exhibit birefringence have directional structure, such as seen in collagen and myelin. Within hearing research, PLM has been used to examine the microcirculation within the guinea pig cochlea , and to study the organization of collagen within the guinea pig  and mouse  basilar membrane after laser irradiation.
Although PLM has proven useful for qualitative comparisons, qPLM has the major advantage of being quantitative. Compared with conventional light microscopy, qPLM provides two additional parameters: retardance (expressed in nanometers) and the average orientation of the polarization axis with the greater index of refraction , henceforth referred to as orientation angle. qPLM has been applied in many clinical specialties, including reproductive biology to quantify spindle aberrations in oocytes , dermatology to improve detection of melanoma , and orthopedics to quantify microstructural remodeling of articular cartilage following defect repair with osteochondral autograft . We were the first to apply qPLM to the inner ear . Specifically, we used qPLM to quantify the polarization dependent optical properties of sectioned cochlear tissues in two different, commonly used mouse models, C57BL/6J and CBA/CaJ. We showed that the networks of collagen fibers known to exist in mammalian cochlea are better-identified using qPLM than using conventional differential interference contrast microscopy.
Encouraged by these results in mice, which are commonly used animal models to investigate hearing and deafness, we undertook the current study to characterize the polarization dependent optical properties of the cadaveric human cochlear sections using qPLM, with an eye to ultimate clinical applications in vivo. We focused on pediatric specimens with no known pathology of the inner ear so to establish a reference for future explorations of pathologic specimens. Many diseases that cause hearing loss are characterized by defects in the quantity, quality and organization of collagen – a major birefringent molecule in the human inner ear [12,13].
2.1 Human cochlear sections
All neonatal human cochleas (n = 79), aged between 0 (term) and 24 months, were reviewed from the Massachusetts Eye and Ear Infirmary collection of the US National Temporal Bone Registry. Twenty one temporal bones reported to have normal labyrinths were selected; the remaining bones were excluded because they had signs of autolysis or otitis media. Of the 21 temporal bones with no documented cochlear histopathology, only 8 had midmodiolar sections with excellent preservation of all cells. These 8 temporal bones were therefore selected for imaging with qPLM. The average time between death and tissue fixation was 8 hours, ranging from 2 hours to 17 hours; 2 cases out of the 8 selected did not have a reported port-mortem time. The study was approved by the Human Studies Committee of the Massachusetts Eye and Ear Infirmary.
2.2 Quantitative polarized microscopy
All archival cochlear specimens studied had been embedded in celloidin, sectioned at 20 μm, and stained with hematoxylin and eosin as per a routine protocol (Merchant and Nadol 2010) in the US temporal bone registry. Section thickness was independently measured and confirmed to be 20 μm using a confocal microscope (Leica TCS SP2 Spectral Confocal Laser Scanning Microscope, Leica Microsystems, Wetzlar, Germany). The sections were mounted onto an inverted microscope (Nikon Eclipse TE 2000-S) and imaged using a commercial birefringence microscopy system (ABRIOTM; Hinds Instruments) to characterize optical anisotropy. This instrument acquires pixwelwise-resolved measurements of retardance and the orientation of the slow optical axis in the specimens, as previously described . In short, monochromatic (wavelength λ = 546 nm) circularly polarized light travels through the specimen and a computer-controlled compensator optic. The change in state of polarization of the light wave is recorded at five different compensator settings, and sample birefringence is calculated based on a polarimetric algorithm, outlined elsewhere . This system resolves retardance values to within 0.02 nm, and it accounts for light absorbance in the sample and a background correction to correct for intrinsic birefringence in the microscope optical elements. The birefringence of the following inner ear structures were measured in each of the four cochlear half turns: organ of Corti, stria vascularis, spiral ligament, basilar membrane, otic capsule and cell bodies of spiral ganglion neurons. The images were analyzed in ImageJ (NIH). Retardance of each cochlear structure was measured three times for each sample to arrive at the mean and standard error of the mean for each structure across different samples. For each cochlear structure with measured retardance, a single factor analysis of variance (ANOVA) was used (in Excel) to test the null hypothesis that the means of retardance for different cochlear turns were all equal; p < 0.05 was considered significant. In addition, a student t-test was used (in Excel) to compare cochlear structures against each other at each different cochlear turn.
Quantitative PLM of the human cochlear sections revealed differences in birefringence across cochlear structures (Figs. 1 and 2), and typically more structural detail than seen under simple light microscopy (Fig. 3). The images in Fig. 1(a) and 2(a) were acquired using a dynamic range from 0 < r < 10 nm, which was larger than any retardance value measured in these samples. The contour scale (0 < r < 4.0 nm) was selected in order to produce sufficient color contrast in the retardance values of the ear structures of interest (e.g. organ of Corti) in the image. Hence, the apparent color saturation shown by the red pixels is a consequence of the selected color scale, not a result of light saturation on the CCD camera. Of the six structures measured, the otic capsule and basilar membrane were the most birefringent (Fig. 4). For all structures, retardance is expressed as mean +/− standard error of the mean. Cochlear turns are analyzed from base to apex because there are well known mechanical, chemical and structural gradients along the length of the spiraling cochlea. Although there was a trend for a decreasing retardance of the otic capsule from the cochlear base toward the apex (Fig. 4) – being 3.89 +/− 0.54 nm in the lower basal turn, 3.75 +/− 0.74 nm in the upper basal turn, 2.78 +/− 0.32 nm in the lower middle turn and 3.02 +/− 0.50 nm in the upper middle turn – this trend was not statistically significant (p = 0.41). The basilar membrane also showed a trend toward decreasing retardance away from the cochlear base (Fig. 4), being 2.25 +/−0.22 nm in the lower basal turn, 2.09 +/− 0.32 nm in the upper basal turn, 1.85 +/− 0.19 nm in the lower middle turn and 1.36 +/− 0.20 nm in the upper middle turn. However, this trend did not meet our criterion for significance (p = 0.07).
Pseudocolor maps of the orientation angle of the birefringent structures are shown in Fig. 1(b) and 2(b). In these images, the color indicates the local orientation of the primary optical axis of the sample consistent with the color wheel shown in the upper right of each image. The cochlear material that exhibited the most regular orientation within the samples was located along the walls of the basal turns and in the basilar membrane, where the orientation angle was aligned tangentially with the perimeter of the wall and the membrane. No consistent orientation in the cochlear material was observed in the otic capsule or the spiral ganglion neurons.
The spiral ligament was the least birefringent structure in the human cochlea (Fig. 4). Differences in retardance across the cochlear half turns were not statistically significant (p = 0.65), being 0.52 +/−0.08 nm in the lower basal turn, 0.56 +/− 0.08 nm in the upper basal turn, 0.46 +/− 0.05 nm in the lower middle turn and 0.47 +/− 0.05 nm in the upper middle turn. Likewise, the retardance of the organ of Corti, which contains sensory hair cells, did not statistically differ across turns (p = 0.99), being 0.78 +/− 0.12 nm in the lower basal turn, 0.76 +/− 0.11 nm in the upper basal, 0.80 +/− 0.16 nm in the lower middle turn and 0.78 +/− 0.13 nm in the upper middle turn. The stria vascularis, a vascular structure that generates the endocochlear potential that drives transduction current through sensory cells, was found to have a greater retardance than the organ of Corti at each half turn. This was found to be statistically significant at the lower and upper basal turn (p = 0.015, p = 0.029) but not at the lower or upper middle turn (p = 0.147, p = 0.116). Retardance of the stria vascularis did not statistically differ between turns (p = 0.64), being 1.35 +/−0.16 nm in the lower basal turn, 1.20 +/− 0.14 nm in the upper basal turn, 1.11 +/− 0.11 nm in the lower middle turn, 1.12 +/− 0.15 nm in the upper middle turn. Retardance of the spiral ganglion was similar to that of the stria vascularis, and not statistically different across turns (p = 0.49), being 0.97 +/−0.15 nm in the lower basal turn, 1.20 +/− 0.14 nm in the upper basal turn, 1.34 +/− 0.21 nm in the lower middle turn, 1.20 +/− 0.18 nm in the upper middle turn.
The retardance of individual structures was analyzed at each different turn using a t-test. The only statistically significant difference identified was the retardance of the basilar membrane between the upper middle and lower basal turn (p = 0.0101).
To the best of our knowledge, this is the first study to characterize the polarization dependent optical properties of human cochlear structures using qPLM. Our results reveal that the otic capsule and basilar membrane are the most birefringent structures whereas the spiral ligament is the least birefringent cochlear structure. While the basilar membrane and the otic capsule tended to have decreasing retardance with distance away from the cochlear base – unlike the more uniform retardance of the organ of Corti, stria vascularis, spiral ligament and spiral ganglion neurons along the cochlear length – this trend did not meet our criterion for significance. The results of this study are comparable with our findings in mice . The level of retardance measured in the human otic capsule was similar to that of the mouse otic capsule of different strains. However, retardance of spiral ganglion cell bodies was notably higher in humans than mice. The greatest difference between human and mouse cochlear section was in retardance of the spiral ligament, which was substantially smaller in humans than in mice. There are several possible reasons for this difference. First, mice are higher-frequency hearing animals – they can hear up to 70 kHz and have maximal sensitivity at 16 kHz whereas humans hear up to 20 kHz with the maximal sensitivity at 2 kHz. Studies in bats, which can hear up to 200 kHz, have suggested that the intricately organized collagen fibers within the spiral ligament that attach to the basilar membrane contribute to the tension in the mechanically-tuned basilar membrane, hence enabling high frequency hearing . Second, the time between death and cochlear extraction is substantially longer for human specimens, averaging 8 hours in our series, compared to just minutes in mice. The prolonged post-mortem time in human specimens may contribute to collagen breakdown and tissue disorganization. In addition, due to the archival nature of human specimens, they were imaged years and decades after cochlear fixation whereas mouse specimens were imaged within days of cochlear extraction; continued tissue breakdown is possible even for fixed specimens. Although our specimens were from young pediatric patients, it is relevant that the human spiral ligament begins to lose collagen-producing fibrocytes early, within the first decade of life , and that degeneration of the spiral ligament occurs at a faster rate than that of other cochlear structures . In addition, fibrocytes of the spiral ligament are particularly sensitive to noise trauma and mild exposure to noise, such as seen in neonatal intensive care units, can cause hearing damage undetectable by threshold based audiogram .
Establishing the polarization dependent optical properties of normal healthy cochlear tissue is the first step in considering qPLM as a diagnostic tool in vivo. Although the histological sections used in this study were fixed, they are likely representative of unfixed tissue, based on our comparison of polarization dependent optical properties of fixed and unfixed cochlear tissues using two photon microscopy . However, we recognize that fixation can change retardance, scattering and depolarization of polarized light , which strongly motivates future studies of unfixed cochlear tissues. Nonetheless, by establishing a normal baseline, our study enables future interpretation of pathologic human specimens, and classification of pathologies that are reflected in abnormal qPLM signatures. Many human diseases are characterized by disorganization or malfunction of collagen in the inner ear , including Alport’s disease , otosclerosis, osteogenesis imperfect and Paget’s disease.
Our study indicates that qPLM is capable of providing quantitative details of human cochlear structures, in particular the organization of collagen networks. Future studies are needed to explore whether application of qPLM in vivo could detect fine pathologic changes early, thus enabling early treatment and possible prevention of hearing loss. Combining qPLM with optical coherence tomography (OCT) may be particularly promising because OCT can generate cross-sectional images with a high spatial resolution based on back-scattered light from a focused beam of infrared light directed at the tissue . The resolution of OCT can range from 2 to 15 μm , which has been sufficient to visualize the microanatomy of the rat cochlea ex vivo [22, 23] and the mouse cochlea in vivo , revealing the three cochlear fluid-filled spaces, modiolus, spiral ligament, the organ of Corti and spiral limbus.
Polarization-sensitive OCT (PS-OCT) is an extension of the conventional OCT, thus allowing measurement of the birefringence and the orientation of the fast axis of a specimen. This generates a quantitative 3D cross sectional image of a specific region of interest. PS-OCT has allowed identification of pathological microstructural changes in the concentration and organization of various tissue types such as tendons and ligaments in the knee [25, 26], and vocal folds of the larynx . PS-OCT has been used during laryngeal surgery to provide immediate feedback , and identify scar tissue within vocal folds to enable precise treatment of the scar tissue that contributes to dysphonia . It would be important to explore whether PS-OCT could similarly generate 3D cross sectional images of the inner ear in vivo, hence providing diagnostic information and invaluable real-time feedback during inner-ear surgery. Such feedback is currently not available but may be crucial in preventing microstructural damage and the associated hearing loss during procedures such as cochlear implantation, stapedectomy or labyrinthectomy. PS-OCT may also enable precise delivery of emerging cell-based and genetic therapies to the diseased regions of the inner ear, and quantitative monitoring of the response to treatment.
A limitation of our study is that it is based on healthy neonatal cochlear sections so that further research is required to explore birefringent patterns in other age groups and in pathological states. Nonetheless, our study highlights the utility of qPLM in visualizing human cochlear structures, and suggests that PS-OCT may be a useful tool to investigate the inner ear and guide precise therapy.
This study provides the first characterization of the polarization dependent optical properties of the microstructures within the human cochlea using qPLM. We note many similarities between the retardance of the human and mouse cochlear tissues, with the notable exceptions of the spiral ligament and spiral ganglion cell bodies, which are much less or more birefringent in humans than in mice, respectively. Taken together, this suggests that techniques developed in mice will have quantitative utility in humans. Our measurements of normal human tissue motivate future exploration of qPLM in diseased tissue associated with hearing loss in order to determine whether birefringence can serve as an imaging biomarker for inner ear diseases that are currently lumped under the umbrella term “sensorineural hearing loss”. Our results, combined with others’ reports of intracochlear imaging using OCT in rodent models, suggest that PS-OCT may have multiple applications in micro-structural diagnosis and targeted therapy of disorders of the human inner ear, while providing real-time feedback during surgical intervention.
We are grateful for support by NIDCD grant K08DC010419 (K.M.S.), the Bertarelli Foundation (K.M.S.), and Curing Kids Foundation at Massachusetts Eye and Ear Infirmary (K.M.S.).
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