Extracellular matrix deformations of the porcine recurrent laryngeal nerve in response to hydrostatic pressure.

Damage to the recurrent laryngeal nerve (RLN) caused by supraphysiological compression or tension imposed by adjacent tissue structures, such as the aorta, may contribute to onset of idiopathic unilateral vocal fold paralysis (iUVP) resulting in difficulty speaking, breathing, and swallowing. We previously demonstrated in adolescent pigs that the right RLN epineurium exhibits uniform composition of adipose tissue, with larger quantities along its length within the neck region in contrast to the left RLN that shows greater collagen composition in the thoracic region and greater quantities of adipose tissue in the neck region. In contrast, the epineurium in piglets was primarily composed of collagen tissue that remained uniform along the length of the left and right RLNs. Tensile testing of the left and right RLN in piglets and pigs showed associated differences in strain by RLN side and segment by age. The goal of this study was to investigate how external hydrostatic compression of the RLN affects the nerve's connective tissue and microstructure. RLN segments were harvested from the distal (cervical/neck) regions and proximal (subclavian for the right RLN, thoracic for the left RLN) regions from eight adolescent pigs and nine piglets. RLN segments were isolated and assessed under fluid compression to test hypotheses regarding epineurium composition and response to applied forces. Second harmonic generation (SHG) imaging of epineurial collagen was conducted at 0, 40, and 80 mmHg of compression. The cartesian strain tensor, principal strain (Eps1), and principal direction of the RLN collagen fibers were determined at each pressure step. Significantly larger values of the 1st principal strain occurred in the proximal segments of the pig left RLN when compared to the same segment in piglets (p = 0.001, pig = 0.0287 [IQR = 0.0161 - 0.0428], piglet = 0.0061 [IQR = 0.0033 - 0.0156]). Additionally, the median transverse strain Eyy) for the second pressure increment was larger in the right proximal segment of pigs compared to piglets (p < 0.001, pig = 0.0122 [IQR = 0.0033 - 0.0171], piglet = 0.0013 [IQR = 0.00001 - 0.0028]). Eyy values were significantly larger in the right proximal RLN versus the left proximal RLNs in pigs but not in piglets (p < 0.001). In contrast to piglets, histological analysis of pig RLN demonstrated increased axial alignment of epineurial and endoneurial collagen in response to compressive pressure. These findings support the hypothesis that the biomechanical response of the RLN to compressive pressure changed from being similar to being different between the right and left RLNs during development in the porcine model. Further investigation of these findings associated with age-related onset of idiopathic UVP may illuminate underlying etiologic mechanisms. STATEMENT OF SIGNIFICANCE: Damage to the recurrent laryngeal nerve (RLN) caused by compression imposed by the aorta may contribute to the onset of left-sided idiopathic unilateral vocal fold paralysis resulting in difficulty speaking, breathing, and swallowing. The goal of this study was to investigate how compression affects the connective tissue and microstructure of the RLN. We quantified the pressure induced deformation of the RLN using multiphoton imaging as a function of both location (proximal versus distal) and age (piglets, adolescent pigs). Our results demonstrate that the biomechanical response of the RLN to compression changes in the right versus left RLN throughout development, providing further evidence that the the left RLN is exposed to increasing dynamic loads with age.

Microstructural Deformations Within the Depth of the Lamina Cribrosa in Response to Acute In Vivo Intraocular Pressure Modulation.

Purpose: The lamina cribrosa (LC) is a leading target for initial glaucomatous damage. We investigated the in vivo microstructural deformation within the LC volume in response to acute IOP modulation while maintaining fixed intracranial pressure (ICP). Methods: In vivo optic nerve head (ONH) spectral-domain optical coherence tomography (OCT) scans (Leica, Chicago, IL, USA) were obtained from eight eyes of healthy adult rhesus macaques (7 animals; ages = 7.9-14.4 years) in different IOP settings and fixed ICP (8-12 mm Hg). IOP and ICP were controlled by cannulation of the anterior chamber and the lateral ventricle of the brain, respectively, connected to a gravity-controlled reservoir. ONH images were acquired at baseline IOP, 30 mm Hg (H1-IOP), and 40 to 50 mm Hg (H2-IOP). Scans were registered in 3D, and LC microstructure measurements were obtained from shared regions and depths. Results: Only half of the eyes exhibited LC beam-to-pore ratio (BPR) and microstructure deformations. The maximal BPR change location within the LC volume varied between eyes. BPR deformer eyes had a significantly higher baseline connective tissue volume fraction (CTVF) and lower pore aspect ratio (P = 0.03 and P = 0.04, respectively) compared to BPR non-deformer. In all eyes, the magnitude of BPR changes in the anterior surface was significantly different (either larger or smaller) from the maximal change within the LC (H1-IOP: P = 0.02 and H2-IOP: P = 0.004). Conclusions: The LC deforms unevenly throughout its depth in response to IOP modulation at fixed ICP. Therefore, analysis of merely the anterior LC surface microstructure will not fully capture the microstructure deformations within the LC. BPR deformer eyes have higher CTVF than BPR non-deformer eyes.

A Subdomain Method for Mapping the Heterogeneous Mechanical Properties of the Human Posterior Sclera.

Although strongly correlated with elevated intraocular pressure, primary open-angle glaucoma (POAG) occurs in normotensive eyes. Mechanical properties of the sclera around the optic nerve head (ONH) may play a role in this disparity. The purpose of this study is to present an automated inverse mechanics based approach to determine the distribution of heterogeneous mechanical properties of the human sclera as derived from its surface deformations arising from pressure inflation experiments. The scleral shell of a 78 year old European Descent male donor eye was utilized to demonstrate the method; the sclera was coated with a speckle pattern on the outer surface and was subjected to inflation pressures of 5, 15, 30, and 45 mmHg. The speckle pattern was imaged at each pressure, and a displacement field was calculated for each pressure step using a previously described sequential digital image correlation (S-DIC) technique. The fiber splay and fiber orientation of the sclera collagen were determined experimentally, and the thickness across the scleral globe was determined using micro CT images. The displacement field from the inflation test was used to calculate the strain and also used as an input for inverse mechanics to determine the heterogeneity of material properties. The scleral geometry was divided into subdomains using the first principal strain. The Holzapfel anisotropic material parameters of matrix and fiber stiffness were estimated within each individual subdomain using an inverse mechanics approach by minimizing the sum of the square of the residuals between the computational and experimental displacement fields. The mean and maximum error in displacement across all subdomains were 8.9 ± 3.0 µm and 13.2 µm, respectively. The full pressure-inflation forward mechanics experiment was done using subdomain-specific mechanical properties on the entire scleral surface. The proposed approach is effective in determining the distribution of heterogeneous mechanical properties of the human sclera in a user-independent manner. Our research group is currently utilizing this approach to better elucidate how scleral stiffness influences those at high risk for POAG.

Racioethnic differences in the biomechanical response of the lamina cribrosa.

Glaucoma is the second leading cause of irreversible blindness in the world with a higher prevalence in those of African Descent (AD) and Hispanic Ethnicity (HE) than in those of European Descent (ED). The objective of this study was to investigate the pressure dependent biomechanical response of the lamina cribrosa (LC) in normal human donor tissues from these racioethnic backgrounds. Pressure inflation tests were performed on 24 human LCs (n = 9 AD, n = 6 ED, and n = 9 HE) capturing the second harmonic generation (SHG) signal of collagen at 5, 15, 30, and 45 mmHg from an anterior view. A non-rigid image registration technique was utilized to determine the 3D displacement field in each LC from which 3D Green strains were calculated. The peak shear strain in the superior quadrant of the LC in those of ED was significantly higher than in those of AD and HE (p-value = 0.005 & 0.034, respectively) where ED = 0.017 [IQR = 0.012-0.027], AD = 0.0002 [IQR = -0.001-0.007], HE = 0.0016 [IQR = -0.002-0.012]). There were also significant differences in the regional strain heterogeneity in those of AD and HE that were absent in those of ED. This work represents, to our knowledge, the first ex-vivo study identifying significant differences in the biomechanical response of the LC in populations at increased risk of glaucoma. Future work will be necessary to assess if and how these differences play a role in predisposing those of Hispanic Ethnicity and African Descent to the onset and/or progression of primary open angle glaucoma. STATEMENT OF SIGNIFICANCE: Glaucoma is the second leading cause of irreversible blindness in the world and occurs more frequently in those of African Descent and Hispanic Ethnicity than in those of European Descent. To date, there has been no ex-vivo study quantifying differences in the biomechanical response of the non-glaucomatous lamina cribrosa (LC) across these racioethnic backgrounds. In this work we report, for the first time, differences in the pressure dependent biomechanical response of LC across different racioethnic groups as quantified using nonlinear optical microscopy. This study lays the foundation for future work investigating if and how these differences may play a role in predisposing those at increased risk to the onset and/or progression of primary open angle glaucoma.