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

Abstract

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.

Figure 1

Characterization of novel organotypic slice NIC model for studying synaptic function following brain injury. (a) Image of simultaneous recordings of injury force and excitatory responses in CA1 hippocampal pyramidal neurons (bottom). (b) Live imaging just prior (top) and at exact time of injury (bottom) at 4× (left) and 40× (right) magnification. (c) Representative recording trace and (d) mean critical pressure (P_c) required to evoke an NIC injury event. (e) Image of injury site after NIC pipette removal (left) labeled with rhodium beads from injury pipette (right). Boxplot represents median and inner and outer (error bars) quartile ranges and is displayed with individual datapoints.

Figure 2

Decrease in glutamate release post-NIC. (a) Representative cell (left) with lucifer yellow (right) in patch pipette for visual identification of a CA1 pyramidal neuron and a representative trace of sEPSCs before and immediately following NIC injury (bottom). (b) Histogram in 10 s bins of average sEPSC events before, during, and after NIC. (c) Average sEPSC frequency (F_(2,16) = 8.39, p = 0.003), (d) frequency expressed as fold change (F_(2,16) = 7.67, p = 0.005), and (e) amplitude (F_(2,16) = 1.15, p = 0.34) before, immediately after, and 5–7 min post-NIC (n = 9 cells). Error bars represent ± SEM. One-way repeated measures ANOVA with Tukey post hoc analysis. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 3

CB1R blockade inhibits transient decreases in synaptic transmission following NIC injury in the hippocampus. (a) Representative trace of sEPSC recording in the presence of CB1 antagonist AM4113 before and immediately post-NIC injury. (b) Histogram in 10 s bins of average sEPSC events with bath application of CB1R antagonist AM4113 (100 nM) before, during, and after NIC. (c) Average sEPSC frequency (F_(2,10) = 1.81, p = 0.213), (d) frequency expressed as fold change (F_(2,10) = 0.366, p = 0.702), and (e) amplitude (F_(2,10) = 1.62, p = 0.246) before, immediately after, and 5–7 min post-NIC (n = 6 cells). Error bars represent ± SEM. One-way repeated measures ANOVA.

Figure 4

Astrocyte activation in NIC-injured brain slices. Representative astrocyte activation (GFAP, red) of (a) control and (b) NIC-injured brain slices at day 0. Representative images of astrocyte activation (GFAP, white) in the hippocampus (white arrows) injured region of brain slices at (c) day 0 (D0) and (d) day 3 (D3) postinjury. Scale bars represent 20 μm in (a and b) and 500 μm in (c and d). Error bars represent ± SEM. Two-way repeated measures ANOVA. ^∗∗p < 0.01.

Figure 5

Day 0 vs. day 3 protein staining. Sham, injury, and injury + antagonist immunohistochemistry staining images for connective tissue growth factor (CTGF, green), tenascin-C (TNC, orange), GFP (red), and merged at day 0 vs. day 3. Scale bar: 50 μm. Scale bars represent 50 μm. Error bars represent ± SEM. Two-way repeated measures ANOVA. ^∗p < 0.05, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.