Simultaneous Quantification of Living Brain Mechanics and Astrocyte Response to Traumatic Brain Injury

Introduction: Mechanics play an integral role in the development and function of biological systems. During development, disease progression, and injury, cells modify their ECM resulting in mechanics. Therefore, methods to measure ECM changes are necessary in understanding the progression of diseases like cancer metastasis and in remodeling after injury. The heterogeneous, complex nature of soft tissues make them especially difficult to characterize. Needle-induced cavitation (NIC) is a technique that uses the principles of cavitation to characterize soft materials at precise locations. Experimental parameters such as needle size and fluid flowrate can tune length and time scales, making it uniquely capable of measuring local mechanical properties of both intact tissue and brain slices. Additionally, NIC has the capability to induce small scale injuries in brain tissue that are associated with impact and blast wave exposure. The simple setup, straightforward measurements, and portability make NIC a particularly appealing characterization technique and allow real time recording of synaptic function post injury.

Materials and Methods: We collected fresh murine brains from 4–20-week-old ex vivo male and female Balb/c mice and C57 black MPY mice. For modulus and fracture energy calculations, NIC was conducted on excised, intact murine brain. A 27 G beveled needle was inserted at precise locations within the tissue and 1X HBSS was pressurized at 250 µL/min until fracture occurred, with pressure monitored over time with a pressure sensor. The fracture energy was calculated using plain strain and axisymmetric hydraulic fracture models. Fracture visualization was confirmed using confocal imaging. Fracture energy measurements using NIC (250 µL/min) and hydraulic fracture models and with pure shear tests (100 µm/s) were conducted on well characterized hard and soft alginate gels (3 and 2.5 vol%). Slice NIC was conducted (5 µL/min) by inserting a ~5 µm diameter glass needle into the hippocampus region of brain visualized using microscopy on 300 µm thick slices. Recording pipettes (3-5MW) were filled with a K+ based internal solution. The GABAA antagonist bicuculline (30µM) and Na+ channel blocker tetrodotoxin (1µM) were added to aCSF to isolate local excitatory postsynaptic currents (mEPSC; VHold=-70mV).

Results and Discussion: From intact brain NIC data, I calculated the modulus of brain regions: cortex 3.6 ± 1.1 kPa, thalamus 5.3 ± 1.4 kPa, hypothalamus 6.4 ± 1 kPa, and cerebellum 3.3 ± 1 kPa (Fig. 1a) and fracture energies: cortex 4.5 ± 1.5 kPa, thalamus 7 ± 1.5 kPa, hypothalamus 7.9 ± 1 kPa, and cerebellum 5 ± 1 kPa (Fig. 1b). To validate my fracture energy results, I prepared hard and soft alginate gels for NIC and pure shear testing, which resulted in moduli of 17.8 ± 7.6 kPa (hard) and 2.9 ± 0.8 kPa (soft), respectively. The fracture energies were consistent between NIC and pure shear: 0.3 ± 0.1 J/m2 (NIC) and 0.2 ± 0.2 J/m2 (pure shear) for soft alginate gels, and 4.3 ± 1.7 J/m2 (NIC) and 4.2 ± 1.0 J/m2 (pure shear) for hard gels. In slice NIC experiments, synaptic function is normal prior to injury, however at the point of NIC injury, a lapse in hippocampal activity occurs for ~2 minutes post-injury (Fig. 1c). After the lapse, the neuron recovers to normal and up to 2-fold higher synaptic activity as indicated by excitatory postsynaptic events (Fig. 1d).

Conclusions: Mechanics of soft tissues that play a role in injury and medical applications. The heterogeneous nature of soft tissues makes them susceptible to fracture or tearing along interfaces at forces associated with impact and blast wave exposure. NIC is a novel technique to measure the fracture energy of intact soft tissues. Quantifying the forces necessary to fracture soft tissue is critical for understanding how impact and blast waves damage tissue in vivo. NIC provides a valuable method to study real time neuronal response to small scale injuries that can elucidate the acute sub-concussive pathways involved in mild traumatic brain injury.