Effects of Mild Closed-Head Injury and Subanesthetic Ketamine Infusion on Microglia, Axonal Injury, and Synaptic Density in Sprague-Dawley Rats

Abstract

Mild traumatic brain injury (mTBI) affects millions of people in the U.S. Approximately 20-30% of those individuals develop adverse symptoms lasting at least 3 months. In a rat mTBI study, the closed-head impact model of engineered rotational acceleration (CHIMERA) produced significant axonal injury in the optic tract (OT), indicating white-matter damage. Because retinal ganglion cells project to the lateral geniculate nucleus (LGN) in the thalamus through the OT, we hypothesized that synaptic density may be reduced in the LGN of rats following CHIMERA injury. A modified SEQUIN (synaptic evaluation and quantification by imaging nanostructure) method, combined with immunofluorescent double-labeling of pre-synaptic (synapsin) and post-synaptic (PSD-95) markers, was used to quantify synaptic density in the LGN. Microglial activation at the CHIMERA injury site was determined using Iba-1 immunohistochemistry. Additionally, the effects of ketamine, a potential neuroprotective drug, were evaluated in CHIMERA-induced mTBI. A single-session repetitive (ssr-) CHIMERA (3 impacts, 1.5 joule/impact) produced mild effects on microglial activation at the injury site, which was significantly enhanced by post-injury intravenous ketamine (10 mg/kg) infusion. However, ssr-CHIMERA did not alter synaptic density in the LGN, although ketamine produced a trend of reduction in synaptic density at post-injury day 4. Further research is necessary to characterize the effects of ssr-CHIMERA and subanesthetic doses of intravenous ketamine on different brain regions and multiple time points post-injury. The current study demonstrates the utility of the ssr-CHIMERA as a rodent model of mTBI, which researchers can use to identify biological mechanisms of mTBI and to develop improved treatment strategies for individuals suffering from head trauma.

Keywords: axonal injury; ketamine; lateral geniculate nucleus; mild traumatic brain injury; rats; synaptic density.

Figure 1

The overall study design and brain tissue analysis. (A): The timeline of the experiment including ssr-CHIMERA, IV ketamine infusion, and brain tissue collection for microglia, axonal injury, and synaptic density analyses. (B): CHIMERA injury site on the cerebral cortex (above the bregma) and examples of activated and inactive microglia. (C): Illustration of axonal injury in the OT (white-matter damage). (D): Synaptic density analysis using SEQUIN with pre-synaptic (synapsin) and post-synaptic (PSD-95) markers, shown as spots (ovals) and puncta (small circles). Synapses are defined as a pair of pre- and post-synaptic puncta separated by <0.55 μm.

Figure 2

Axonal injury in the OT following ssr-CHIMERA injury in rats. (A): Representative of silver staining images in the OT (stereotaxic coordinates, AP: −2.92 mm, ML: ±3.4 mm, DV: 8.4 mm from the bregma), taken at 4× magnification. (B): CHIMERA injury significantly increased axonal injury in the OT compared to the sham group (N = 10 per group, * p < 0.05). Reproduced from the previous study with permission [7].

Figure 3

Microglial activation on the cortical injury site (primary motor cortex and somatosensory cortex) following ssr-CHIMERA in rats. (A): A representative image of the cortical injury site (arrow) in a CHIMERA-saline animal (10×). (B): A representative image of cortical injury site (arrow) in a CHIMERA-ketamine animal (10×). (C): A representative image of inactive (ramified) microglia (40×). (D): A representative image of activated (ameboid) microglia (40×). (E): Increased microglial activation in the CHIMERA-ketamine group compared to the CHIMERA-saline group (N = 6 per group, * p < 0.05).

Figure 4

The SEQUIN analysis for synaptic density in the LGN. (A): The LGN (in yellow) depicted in the rat brain atlas (AP: −3.72 mm, ML: ±3.5 mm, DV: 4.6 mm from the bregma). (B): A confocal microscope image of synapsin (green), PSD-95 (red), and synaptic puncta (yellow) in the LGN. (C): A processed image of spot analysis using Imaris software version 10.0.1. (D): Ketamine infusion reduced synapsin density in the LGN. (E): No significant effects of CHIMERA or ketamine on PSD-95 density in the LGN. (F): No significant effects of CHIMERA or ketamine on synaptic density in the LGN. (N = 9–10 per group, * p < 0.05).