Needle-induced cavitation: A method to probe the local mechanics of brain tissue.

Traditional mechanical characterization of extremely soft tissues is challenging given difficulty extracting tissue, satisfying geometric requirements, keeping tissues hydrated, and securing the tissue in an apparatus without slippage. The heterogeneous nature and structural complexity of brain tissues on small length scales makes it especially difficult to characterize. Needle-induced cavitation (NIC) is a technique that overcomes these issues and can mechanically characterize brain tissues at precise, micrometer-scale locations. This small-scale capability is crucial in order to spatially characterize diseased tissue states like fibrosis or cancer. NIC consists of inserting a needle into a tissue and pressurizing a fluid until a deformation occurs at the tip of the needle at a critical pressure. NIC is a convenient, affordable technique to measure mechanical properties, such as modulus and fracture energy, and to assess the performance of soft materials. Experimental parameters such as needle size and fluid flowrate are tunable, so that the end-user can control the length and time scales, making it uniquely capable of measuring local mechanical properties across a wide range of strain rates. The portable nature of NIC and capability to conduct in vivo experiments makes it a particularly appealing characterization technique compared to traditional methods. Despite significant developments in the technique over the last decade, wide implementation in the biological field is still limited. Here, we address the limitations of the NIC technique specifically when working with soft tissues and provide readers with expected results for brain tissue. Our goal is to assist others in conducting reliable and reproducible mechanical characterization of soft biomaterials and tissues.

Short-term neural and glial response to mild traumatic brain injury in the hippocampus.

Traumatic brain injury (TBI) is an established risk factor for developing neurodegenerative disease. However, how TBI leads from acute injury to chronic neurodegeneration is limited to postmortem models. There is a lack of connections between in vitro and in vivo TBI models that can relate injury forces to both macroscale tissue damage and brain function at the cellular level. Needle-induced cavitation (NIC) is a technique that can produce small cavitation bubbles in soft tissues, which allows us to relate small strains and strain rates in living tissue to ensuing acute cell death, tissue damage, and tissue remodeling. Here, we applied NIC to mouse brain slices to create a new model of TBI with high spatial and temporal resolution. We specifically targeted the hippocampus, which is a brain region critical for learning and memory and an area in which injury causes cognitive pathologies in humans and rodent models. By combining NIC with patch-clamp electrophysiology, we demonstrate that NIC in the cornu ammonis 3 region of the hippocampus dynamically alters synaptic release onto cornu ammonis 1 pyramidal neurons in a cannabinoid 1 receptor-dependent manner. Further, we show that NIC induces an increase in extracellular matrix protein GFAP associated with neural repair that is mitigated by cannabinoid 1 receptor antagonism. Together, these data lay the groundwork for advanced approaches in understanding how TBI impacts neural function at the cellular level and the development of treatments that promote neural repair in response to brain injury.

Acute and Chronic Neural and Glial Response to Mild Traumatic Brain Injury in the Hippocampus.

Traumatic brain injury (TBI) is an established risk factor for developing neurodegenerative disease. However, how TBI leads from acute injury to chronic neurodegeneration is limited to post-mortem models. There is a lack of connections between in vitro and in vivo TBI models that can relate injury forces to both macroscale tissue damage and brain function at the cellular level. Needle-induced cavitation (NIC) is a technique that can produce small cavitation bubbles in soft tissues, which allows us to relate small strains and strain rates in living tissue to ensuing acute and chronic cell death, tissue damage, and tissue remodeling. Here, we applied NIC to mouse brain slices to create a new model of TBI with high spatial and temporal resolution. We specifically targeted the hippocampus, which is a brain region critical for learning and memory and an area in which injury causes cognitive pathologies in humans and rodent models. By combining NIC with patch-clamp electrophysiology, we demonstrate that NIC in the Cornu Ammonis (CA)3 region of the hippocampus dynamically alters synaptic release onto CA1 pyramidal neurons in a cannabinoid 1 receptor (CB1R)-dependent manner. Further, we show that NIC induces an increase in extracellular matrix proteins associated with neural repair that is mitigated by CB1R antagonism. Together, these data lay the groundwork for advanced approaches in understanding how TBI impacts neural function at the cellular level, and the development of treatments that promote neural repair in response to brain injury.

Tenascin-C Activation of Lung Fibroblasts in a 3D Synthetic Lung Extracellular Matrix Mimic.

The lung extracellular matrix (ECM) maintains the structural integrity of the tissue and regulates the phenotype and functions of resident fibroblasts. Lung-metastatic breast cancer alters these cell-ECM interactions, promoting fibroblast activation. There is a need for bio-instructive ECM models that match the ECM composition and biomechanics of the lung to study these cell-matrix interactions in vitro. Here, a synthetic, bioactive hydrogel is synthesized that mimics the native lung modulus and includes a representative distribution of the most abundant ECM peptide motifs responsible for integrin-binding and matrix metalloproteinase (MMP)-mediated degradation in the lung, which enables quiescent culture of human lung fibroblasts (HLFs). Stimulation with transforming growth factor ß1 (TGF-ß1), metastatic breast cancer conditioned media (CM), or tenascin-C-derived integrin-binding peptide activated hydrogel-encapsulated HLFs demonstrates multiple environmental methods to activate HLFs in a lung ECM-mimicking hydrogel. This lung hydrogel platform is a tunable, synthetic approach to studying the independent and combinatorial effects of ECM in regulating fibroblast quiescence and activation.

Tenascin-C activation of lung fibroblasts in a 3D synthetic lung extracellular matrix mimic.

The lung extracellular matrix (ECM) maintains the structural integrity of the tissue and regulates the phenotype and functions of resident fibroblasts. Lung-metastatic breast cancer alters these cell-ECM interactions, promoting fibroblast activation. There is a need for bio-instructive ECM models that contain the ECM composition and biomechanics of the lung to study these cell-matrix interactions in vitro . Here, we developed a synthetic, bioactive hydrogel that mimics the native lung modulus, and includes a representative distribution of the most abundant ECM peptide motifs responsible for integrin binding and matrix metalloproteinase (MMP)-mediated degradation in the lung, which promotes quiescence of human lung fibroblasts (HLFs). Stimulation with transforming growth factor ß1 (TGF-ß1), metastatic breast cancer conditioned media (CM), or tenascin-C activated these hydrogel-encapsulated HLFs in a manner reflective of their native in vivo responses. We propose this lung hydrogel platform as a tunable, synthetic approach to study the independent and combinatorial effects of ECM in regulating fibroblast quiescence and activation.

Cavitation induced fracture of intact brain tissue.

Nonpenetrating traumatic brain injuries (TBIs) are linked to cavitation. The structural organization of the brain makes it particularly susceptible to tears and fractures from these cavitation events, but limitations in existing characterization methods make it difficult to understand the relationship between fracture and cavitation in this tissue. More broadly, fracture energy is an important, yet often overlooked, mechanical property of all soft tissues. We combined needle-induced cavitation with hydraulic fracture models to induce and quantify fracture in intact brains at precise locations. We report here the first measurements of the fracture energy of intact brain tissue that range from 1.5 to 8.9 J/m2, depending on the location in the brain and the model applied. We observed that fracture consistently occurs along interfaces between regions of brain tissue. These fractures along interfaces allow cavitation-related damage to propagate several millimeters away from the initial injury site. Quantifying the forces necessary to fracture brain and other soft tissues is critical for understanding how impact and blast waves damage tissue in vivo and has implications for the design of protective gear and tissue engineering.

Seeded laser-induced cavitation for studying high-strain-rate irreversible deformation of soft materials.

Characterizing the high-strain-rate and high-strain mechanics of soft materials is critical to understanding the complex behavior of polymers and various dynamic injury mechanisms, including traumatic brain injury. However, their dynamic mechanical deformation under extreme conditions is technically difficult to quantify and often includes irreversible damage. To address such challenges, we investigate an experimental method, which allows quantification of the extreme mechanical properties of soft materials using ultrafast stroboscopic imaging of highly reproducible laser-induced cavitation events. As a reference material, we characterize variably cross-linked polydimethylsiloxane specimens using this method. The consistency of the laser-induced cavitation is achieved through the introduction of laser absorbing seed microspheres. Based on a simplified viscoelastic model, representative high-strain-rate shear moduli and viscosities of the soft specimens are quantified across different degrees of crosslinking. The quantified rheological parameters align well with the time-temperature superposition prediction of dynamic mechanical analysis. The presented method offers significant advantages with regard to quantifying high-strain rate, irreversible mechanical properties of soft materials and tissues, compared to other methods that rely upon the cyclic dynamics of cavitation. These advances are anticipated to aid in the understanding of how damage and injury develop in soft materials and tissues.

Differentiation of Pathogenic Th17 Cells Is Negatively Regulated by Let-7 MicroRNAs in a Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a disabling demyelinating autoimmune disorder of the central nervous system (CNS) which is driven by IL-23- and IL-1ß-induced autoreactive Th17 cells that traffic to the CNS and secrete proinflammatory cytokines. Th17 pathogenicity in MS has been correlated with the dysregulation of microRNA (miRNA) expression, and specific miRNAs have been shown to promote the pathogenic Th17 phenotype. In the present study, we demonstrate, using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), that let-7 miRNAs confer protection against EAE by negatively regulating the proliferation, differentiation and chemokine-mediated migration of pathogenic Th17 cells to the CNS. Specifically, we found that let-7 miRNAs may directly target the cytokine receptors Il1r1 and Il23r, as well as the chemokine receptors Ccr2 and Ccr5. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for disease treatment.

Cross-platform mechanical characterization of lung tissue.

Published data on the mechanical strength and elasticity of lung tissue is widely variable, primarily due to differences in how testing was conducted across individual studies. This makes it extremely difficult to find a benchmark modulus of lung tissue when designing synthetic extracellular matrices (ECMs). To address this issue, we tested tissues from various areas of the lung using multiple characterization techniques, including micro-indentation, small amplitude oscillatory shear (SAOS), uniaxial tension, and cavitation rheology. We report the sample preparation required and data obtainable across these unique but complimentary methods to quantify the modulus of lung tissue. We highlight cavitation rheology as a new method, which can measure the modulus of intact tissue with precise spatial control, and reports a modulus on the length scale of typical tissue heterogeneities. Shear rheology, uniaxial, and indentation testing require heavy sample manipulation and destruction; however, cavitation rheology can be performed in situ across nearly all areas of the lung with minimal preparation. The Young's modulus of bulk lung tissue using micro-indentation (1.4±0.4 kPa), SAOS (3.3±0.5 kPa), uniaxial testing (3.4±0.4 kPa), and cavitation rheology (6.1±1.6 kPa) were within the same order of magnitude, with higher values consistently reported from cavitation, likely due to our ability to keep the tissue intact. Although cavitation rheology does not capture the non-linear strains revealed by uniaxial testing and SAOS, it provides an opportunity to measure mechanical characteristics of lung tissue on a microscale level on intact tissues. Overall, our study demonstrates that each technique has independent benefits, and each technique revealed unique mechanical features of lung tissue that can contribute to a deeper understanding of lung tissue mechanics.