Comparison of the Biomechanics of the Mouse Astrocytic Lamina Cribrosa Between Glaucoma and Optic Nerve Crush Models.

Purpose: The strain response of the mouse astrocytic lamina (AL) to an ex vivo mechanical test was compared between two protocols: eyes that underwent sustained intraocular pressure (IOP) increase and eyes after optic nerve crush. Methods: Chronic IOP elevation was induced by microbead injection or the optic nerve was crushed in mice with widespread green fluorescence. After 3 days or 6 weeks, eyes were inflation tested by a published method of two-photon fluorescence to image the AL. Digital volume correlation was used to calculate strains. Optic nerve axon damage was also evaluated. Results: In the central AL but not the peripheral AL, four strains were greater in eyes at the 3-day glaucoma time point than control (P from 0.029 to 0.049, n = 8 eyes per group). Also, at this time point, five strains were greater in the central AL compared to the peripheral AL (P from 0.041 to 0.00003). At the 6-week glaucoma time point, the strains averaged across the specimen, in the central AL, and the peripheral AL were indistinguishable from the respective controls. Strains were not significantly different between controls and eyes 3 days or 6 weeks after crush (n = 8 and 16). Conclusions: We found alterations in the ex vivo mechanical behavior in eyes from mice with experimental glaucoma but not in those with crushed optic nerves. The results of this study demonstrate that significant axon injury does not directly affect mechanical behavior of the astrocytic lamina.

Computational study of the mechanical behavior of the astrocyte network and axonal compartments in the mouse optic nerve head.

Glaucoma is a blinding disease characterized by the degeneration of the retinal ganglion cell (RGC) axons at the optic nerve head (ONH). A major risk factor for glaucoma is the intraocular pressure (IOP). However, it is currently impossible to measure the IOP-induced mechanical response of the axons of the ONH. The objective of this study was to develop a computational modeling method to estimate the IOP-induced strains and stresses in the axonal compartments in the mouse astrocytic lamina (AL) of the ONH, and to investigate the effect of the structural features on the mechanical behavior. We developed experimentally informed finite element (FE) models of six mouse ALs to investigate the effect of structure on the strain responses of the astrocyte network and axonal compartments to pressure elevation. The specimen-specific geometries of the FE models were reconstructed from confocal fluorescent images of cryosections of the mouse AL acquired in a previous study that measured the structural features of the astrocytic processes and axonal compartments. The displacement fields obtained from digital volume correlation in prior inflation tests of the mouse AL were used to determine the displacement boundary conditions of the FE models. We then applied Gaussian process regression to analyze the effects of the structural features on the strain outcomes simulated for the axonal compartments. The axonal compartments experienced, on average, 6 times higher maximum principal strain but 1800 times lower maximum principal stress compared to those experienced by the astrocyte processes. The strains experienced by the axonal compartments were most sensitive to variations in the area of the axonal compartments. Larger axonal compartments that were more vertically aligned, closer to the AL center, and with lower local actin area fraction had higher strains. Understanding the factors affecting the deformation in the axonal compartments will provide insights into mechanisms of glaucomatous axonal damage.

Decellularization compromises mechanical and structural properties of the native trachea.

Tracheal replacement using tissue engineering technologies offers great potential to improve previously intractable clinical interventions, and interest in this area has increased in recent years. Many engineered airway constructs currently rely on decellularized native tracheas to serve as the scaffold for tissue repair. Yet, mechanical failure leading to airway narrowing and collapse remains a major cause of morbidity and mortality following clinical implantation of decellularized tracheal grafts. To understand better the factors contributing to mechanical failure in vivo, we characterized the histo-mechanical properties of tracheas following two different decellularization protocols, including one that has been used clinically. All decellularized tracheas deviated from native mechanical behavior, which may provide insights into observed in vivo graft failures. We further analyzed protein content by western blot and analyzed microstructure by histological staining and found that the specific method of decellularization resulted in significant differences in the depletion of proteoglycans and degradation of collagens I, II, III, and elastin. Taken together, this work demonstrates that the heterogeneous architecture and mechanical behavior of the trachea is severely compromised by decellularization. Such structural deterioration may contribute to graft failure clinically and limit the potential of decellularized native tracheas as viable long-term orthotopic airway replacements.

Mechanical strain in the mouse astrocytic lamina increases after exposure to recombinant trypsin.

The responses of astrocytes in the optic nerve head (ONH) to mechanical and biochemical stimuli are important to understanding the degeneration of retinal ganglion cell axons in glaucoma. The ONH in glaucoma is vulnerable to stress produced by the intraocular pressure (IOP). Notably, after three days of elevated IOP in a mouse model, the junctions between the astrocytic processes and the peripapillary sclera were altered and the structural compliance of the ONH increased. In order to simulate this aspect of glaucomatous remodeling, explanted mouse eyes were treated with TrypLE, a recombinant trypsin enzyme. Treatment with TrypLE caused the periphery of the astrocytic lamina to contract radially by 0.044 ± 0.038. Transmission electron microscopy showed that TrypLE caused a separation of the end-feet of the astrocyte processes from the basement membrane at the junction with the sclera. Inflation testing after treatment with TrypLE caused an increased strain response in the astrocytic lamina compared to the strain response before treatment. The greatest increase was in the radial Green-Lagrange strain, Err = 0.028 ± 0.009, which increased by 340%. The alterations in the microstructure and in the strain response of the astrocytic lamina reported in mouse experimental glaucoma were partially reproduced by experimental treatment of mouse eyes with TrypLE. The results herein suggest that separation of junctions between the astrocyte processes and the sclera may be instrumental in increasing the structural compliance of the ONH after a period of elevated IOP. STATEMENT OF SIGNIFICANCE: Astrocytes of the optic nerve of the eye spread out from edge to edge across the optic nerve in a region referred to as the astrocytic lamina. In an experimental model of glaucoma caused by elevated eye-pressure, there is disruption of the connections between astrocytes and the edge of the astrocytic lamina. We caused a similar event in the lamina by incubating explanted mouse eyes with an enzyme. Disruption of the astrocyte connections to the edge of their tissue caused the tissue to stretch more when we increased the eye-pressure, compared to the control tissue. This work is the first on the tissue of the optic nerve to demonstrate the importance of cell connections in preventing the over-stretching of the astrocytic lamina.

Effects of experimental glaucoma in Lama1nmf223 mutant mice.

To identify changes in response to experimental intraocular pressure (IOP) elevation associated with the laminin α1 nmf223 mutation in mice. Laminin mutant (LM) mice (Lama1nmf223) and C57BL/6J (B6) mice in two age groups each (4-5 months and >1 year) underwent intracameral microbead injections to produce unilaterally elevated IOP. We assessed axonal transport block of immunofluorescently labeled amyloid precursor protein (APP) after 3 days and retinal ganglion cell (RGC) axon loss after 6 weeks. Light, electron and fluorescent microscopy was used to study baseline anatomic differences and effects of 3-day IOP elevation in younger LM mice. In younger mice of both LM and B6 strains, elevated IOP led to increased APP block in the retina, prelaminar optic nerve head (preONH), unmyelinated optic nerve (UON), and myelinated optic nerve (MON). APP blockade not significantly different between younger B6 and LM mouse strains. Older LM mice had greater APP accumulation in both control and glaucoma eyes compared to older B6, however, accumulation was not significantly greater in LM glaucoma eyes compared to LM controls. Axon loss at 6 weeks was 12.2% in younger LM and 18.7% in younger B6 mice (difference between strains, p = 0.22, Mann Whitney test). Untreated LM optic nerve area was lower compared to B6 (nerve area, p < 0.0001, t-test). Aberrant axon bundles, as well as defects, thickening and reduplication of pia mater, were seen in the optic nerves of younger LM mice. Axonal transport blockade significantly differed between old B6 and old LM mice in control and glaucoma eyes, and younger LM mice had abnormal axon paths and lower optic nerve area.

The role of aquaporin-4 in optic nerve head astrocytes in experimental glaucoma.

PURPOSE: To study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null). METHODS: C57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression, and transport block were also analyzed. RESULTS: B6 mice had abundant AQP4 expression in Müller cells, astrocytes of retina and myelinated ON (MON), but minimal AQP4in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p≤0.05). Bead-injection led to comparable 3D-IOP elevation (p = 0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6). CONCLUSION: Lack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head.

Biomechanics of the optic nerve head and peripapillary sclera in a mouse model of glaucoma.

The deformation of the mouse astrocytic lamina (AL) and adjacent peripapillary sclera (PPS) was measured in response to elevated intraocular pressure. We subjected explanted mouse eyes to inflation testing, comparing control eyes to those 3 days and 6 weeks after induction of ocular hypertension (OHT) via ocular microbead injection. Laser scanning microscopy was used with second harmonic generation to image the collagenous PPS and two-photon fluorescence to image transgenic fluorescent astrocytes in the AL. Digital volume correlation was applied to calculate strains in the PPS and AL. The specimen-averaged strains were biaxial in the AL and PPS, with greater strain overall in the x- than y-direction in the AL and greater strain in the θ- than the r-direction in the PPS. Strains increased after 3-day OHT, with greater strain overall in the 3-day AL than control AL, and greater circumferential strain in the 3-day PPS than control PPS. In the 6-week OHT eyes, AL and PPS strains were similar overall to controls. This experimental glaucoma model demonstrated a dynamic change in the mechanical behaviour of the AL and PPS over time at the site of neuronal injury and remodelling in glaucoma.

Astrocyte responses to experimental glaucoma in mouse optic nerve head.

PURPOSE: To delineate responses of optic nerve head astrocytes to sustained intraocular pressure (IOP) elevation in mice. METHODS: We elevated IOP for 1 day to 6 weeks by intracameral microbead injection in 4 strains of mice. Astrocyte alterations were studied by transmission electron microscopy (TEM) including immunogold molecular localization, and by laser scanning microscopy (LSM) with immunofluorescence for integrin ß1, α-dystroglycan, and glial fibrillary acidic protein (GFAP). Astrocyte proliferation and apoptosis were quantified by Ki67 and TUNEL labeling, respectively. RESULTS: Astrocytes in normal optic nerve head expressed integrin ß1 and α-dystroglycan by LSM and TEM immunogold labeling at electron dense junctional complexes that were found only on cell membrane zones bordering their basement membranes (BM) at the peripapillary sclera (PPS) and optic nerve head capillaries. At 1-3 days after IOP elevation, abnormal extracellular spaces appeared between astrocytes near PPS, and axonal vesical and mitochondrial accumulation indicated axonal transport blockade. By 1 week, abnormal spaces increased, new collagen formation occurred, and astrocytes separated from their BM, leaving cell membrane fragments. Electron dense junctional complexes separated or were absent at the BM. Astrocyte proliferation was modest during the first week, while only occasional apoptotic astrocytes were observed by TEM and TUNEL. CONCLUSIONS: Astrocytes normally exhibit junctions with their BM which are disrupted by extended IOP elevation. Responses include reorientation of cell processes, new collagen formation, and cell proliferation.

A method to quantify regional axonal transport blockade at the optic nerve head after short term intraocular pressure elevation in mice.

Axonal transport blockade is an initial step in retinal ganglion cell (RGC) degeneration in glaucoma and targeting maintenance of normal axonal transport could confer neuroprotection. We present an objective, quantitative method for assessing axonal transport blockade in mouse glaucoma models. Intraocular pressure (IOP) was elevated unilaterally in CD1 mice for 3 days using intracameral microbead injection. Longitudinal sections of optic nerve head (ONH) were immunofluorescently labeled for myelin basic protein (MBP) and amyloid precursor protein (APP), which is transported predominantly orthograde by neurons. The beginning of the myelin transition zone, visualized with the MBP label, was more posterior with elevated IOP, 288.8 ± 40.9 µm, compared to normotensive control eyes, 228.7 ± 32.7 µm (p = 0.030, N = 6 pairs). Glaucomatous regional APP accumulations in retina, prelaminar ONH, unmyelinated ONH, and myelinated optic nerve were identified by objective qualification of pixels with fluorescent intensity greater than the 97.5th percentile value of control eyes (suprathreshold pixels). This method segregated images with APP blockade from those with normal transport of APP. The fraction of suprathreshold pixels was significantly higher following IOP elevation than in normotensive controls in the unmyelinated ONH and myelinated nerve regions (paired analyses, p = 0.02 and 0.003, respectively, N = 12), but not in retina or prelaminar ONH (p = 0.91 and 0.08, respectively). The mean intensity of suprathreshold pixels was also significantly greater in glaucoma than in normotensive controls in prelaminar ONH, unmyelinated ONH and myelinated optic nerve (p = 0.01, 0.01, 0.002, respectively). Using this method, subconjunctival glyceraldehyde, which is known to worsen long-term RGC loss with IOP elevation, also produced greater APP blockade, but not statistically significant compared to glaucoma alone. Systemic losartan, which aids RGC axonal survival in glaucoma, reduced APP blockade, but not statistically significant compared to glaucoma alone. The method provides a short-term assessment of axonal injury for use in initial tests of neuroprotective therapies that may beneficially affect RGC transport in animal models of glaucoma.

Chronic mTOR activation induces a degradative smooth muscle cell phenotype.

Smooth muscle cell (SMC) proliferation has been thought to limit the progression of thoracic aortic aneurysm and dissection (TAAD) because loss of medial cells associates with advanced disease. We investigated effects of SMC proliferation in the aortic media by conditional disruption of Tsc1, which hyperactivates mTOR complex 1. Consequent SMC hyperplasia led to progressive medial degeneration and TAAD. In addition to diminished contractile and synthetic functions, fate-mapped SMCs displayed increased proteolysis, endocytosis, phagocytosis, and lysosomal clearance of extracellular matrix and apoptotic cells. SMCs acquired a limited repertoire of macrophage markers and functions via biogenesis of degradative organelles through an mTOR/ß-catenin/MITF-dependent pathway, but were distinguishable from conventional macrophages by an absence of hematopoietic lineage markers and certain immune effectors even in the context of hyperlipidemia. Similar mTOR activation and induction of a degradative SMC phenotype in a model of mild TAAD due to Fbn1 mutation greatly worsened disease with near-uniform lethality. The finding of increased lysosomal markers in medial SMCs from clinical TAAD specimens with hyperplasia and matrix degradation further supports the concept that proliferation of degradative SMCs within the media causes aortic disease, thus identifying mTOR-dependent phenotypic modulation as a therapeutic target for combating TAAD.

Maladaptive aortic remodeling in hypertension associates with dysfunctional smooth muscle contractility.

Intramural cells are responsible for establishing, maintaining, and restoring the functional capability and structural integrity of the aortic wall. In response to hypertensive loading, these cells tend to increase wall content via extracellular matrix turnover in an attempt to return wall stress and/or material stiffness toward homeostatic values despite the elevated pressure. Using a common rodent model of induced hypertension, we found marked mouse-to-mouse differences in thoracic aortic remodeling over 2-4 wk of pressure elevation, with mechanoadaptation in some but gross maladaptation in most mice despite the same experimental conditions and overall genetic background. Consistent with our hypothesis, we also found a strong correlation between maladaptive aortic remodeling and a dysfunctional ability of the vessel to vasoconstrict, with maladaptation often evidenced by marked adventitial fibrosis. Remarkably, mouse-to-mouse variability did not correlate with the degree or duration of pressure elevation over the 2- to 4-wk study period. These findings suggest both a need to study together the structure, mechanical properties, and function across layers of the wall when assessing aortic health and a need for caution in using common statistical comparisons across small seemingly well-defined groups that may mask important underlying individual responses, an area of investigation that demands increasing attention as we move toward an era of precision diagnosis and patient care. NEW & NOTEWORTHY There are three primary findings. Marked mouse-to-mouse differences exist in large vessel hypertensive remodeling in an otherwise equivalent cohort of animals. The degree of maladaptation correlates strongly with decreases in smooth muscle contractile capacity. Finally, short-term maladaptive remodeling is independent of the precise degree or duration of the pressure elevation provided that thresholds are exceeded. Therapeutic targets should thus be personalized and focus on both layer-to-layer interactions and early interventions.

LNK deficiency promotes acute aortic dissection and rupture.

Aortic dissection (AD) is a life-threatening vascular disease with limited treatment strategies. Here, we show that loss of the GWAS-identified SH2B3 gene, encoding lymphocyte adaptor protein LNK, markedly increases susceptibility to acute AD and rupture in response to angiotensin (Ang) II infusion. As early as day 3 following Ang II infusion, prior to the development of AD, Lnk-/- aortas display altered mechanical properties, increased elastin breaks, collagen thinning, enhanced neutrophil accumulation, and increased MMP-9 activity compared with WT mice. Adoptive transfer of Lnk-/- leukocytes into Rag1-/- mice induces AD and rupture in response to Ang II, demonstrating that LNK deficiency in hematopoietic cells plays a key role in this disease. Interestingly, treatment with doxycycline prevents the early accumulation of aortic neutrophils and significantly reduces the incidence of AD and rupture. PrediXcan analysis in a biobank of more than 23,000 individuals reveals that decreased expression of SH2B3 is significantly associated with increased frequency of AD-related phenotypes (odds ratio 0.81). Thus, we identified a role for LNK in the pathology of AD in experimental animals and humans and describe a new model that can be used to inform both inherited and acquired forms of this disease.

Deficient Circumferential Growth Is the Primary Determinant of Aortic Obstruction Attributable to Partial Elastin Deficiency.

OBJECTIVE: Williams syndrome is characterized by obstructive aortopathy attributable to heterozygous loss of ELN, the gene encoding elastin. Lesions are thought to result primarily from excessive smooth muscle cell (SMC) proliferation and consequent medial expansion, although an initially smaller caliber and increased stiffness of the aorta may contribute to luminal narrowing. The relative contributions of such abnormalities to the obstructive phenotype had not been defined. APPROACH AND RESULTS: We quantified determinants of luminal stenosis in thoracic aortas of Eln-/- mice incompletely rescued by human ELN. Moderate obstruction was largely because of deficient circumferential growth, most prominently of ascending segments, despite increased axial growth. Medial thickening was evident in these smaller diameter elastin-deficient aortas, with medial area similar to that of larger diameter control aortas. There was no difference in cross-sectional SMC number between mutant and wild-type genotypes at multiple stages of postnatal development. Decreased elastin content was associated with medial fibrosis and reduced aortic distensibility because of increased structural stiffness but preserved material stiffness. Elastin-deficient SMCs exhibited greater contractile-to-proliferative phenotypic modulation in vitro than in vivo. We confirmed increased medial collagen without evidence of increased medial area or SMC number in a small ascending aorta with thickened media of a Williams syndrome subject. CONCLUSIONS: Deficient circumferential growth is the predominant mechanism for moderate obstructive aortic disease resulting from partial elastin deficiency. Our findings suggest that diverse aortic manifestations in Williams syndrome result from graded elastin content, and SMC hyperplasia causing medial expansion requires additional elastin loss superimposed on ELN haploinsufficiency.