Concussion Induces Hippocampal Circuitry Disruption in Swine

Abstract

Hippocampal-dependent deficits in learning and memory formation are a prominent feature of traumatic brain injury (TBI); however, the role of the hippocampus in cognitive dysfunction after concussion (mild TBI) is unknown. We therefore investigated functional and structural changes in the swine hippocampus following TBI using a model of head rotational acceleration that closely replicates the biomechanics and neuropathology of closed-head TBI in humans. We examined neurophysiological changes using a novel ex vivo hippocampal slice paradigm with extracellular stimulation and recording in the dentate gyrus and CA1 occurring at 7 days following non-impact inertial TBI in swine. Hippocampal neurophysiology post-injury revealed reduced axonal function, synaptic dysfunction, and regional hyperexcitability at one week following even "mild" injury levels. Moreover, these neurophysiological changes occurred in the apparent absence of intra-hippocampal neuronal or axonal degeneration. Input-output curves demonstrated an elevated excitatory post-synaptic potential (EPSP) output for a given fiber volley input in injured versus sham animals, suggesting a form of homeostatic plasticity that manifested as a compensatory response to decreased axonal function in post-synaptic regions. These data indicate that closed-head rotational acceleration-induced TBI, the common cause of concussion in humans, may induce significant alterations in hippocampal circuitry function that have not resolved at 7 days post-injury. This circuitry dysfunction may underlie some of the post-concussion symptomatology associated with the hippocampus, such as post-traumatic amnesia and ongoing cognitive deficits.

Keywords: axonal pathology; concussion; epileptogenesis; hippocampus; mild TBI; traumatic brain injury.

FIG. 1.

Hippocampal slice recordings following rotational TBI in swine. Closed-head diffuse brain injury was induced based on rapid rotational acceleration-deceleration in the coronal plane as depicted in (A). At terminal time-points of 7 days post-injury, pigs were transcardially perfused with cold, oxygenated artificial cerebrospinal fluid, the posterior left quadrant of the brain was removed (B), and the remainder of the brain was fixed with paraformaldehyde. The live posterior quadrant was immediately dissected on a cold block, and the hippocampus was removed and blocked transversely to the long axis. Approximately 1cm of the dorsal hippocampus was then transferred to sucrose cutting solution (see Methods) and then 350 μM sections were cut on a vibratome. These sections were transferred to an interface chamber (C), where stimulation (red circles) and recordings (black arrows) (D) were performed in the Schaffer collaterals (CA1) and the medial and lateral perforant path (dentate). A typical trace from CA1 (E) had a prominent fiber volley (FV) (arrow) immediately following the stimulation (artifact removed, *) followed by an excitatory post-synaptic potential (EPSP). (D) Modified from http://anatomie.vetmed.uni-leipzig.de/external/hippocampus/hippocampus_schema_gross.jpg

FIG. 2.

Changes in response waveform post-TBI in area CA1 and dentate gyrus. Representative examples of responses to stimuli in the Schaffer collaterals (CA1) or medial perforant path (mPP) and lateral performant path (lPP) in hippocampal slices after rotational brain injury in swine. Each waveform is the average of 10 traces at the half-maximal stimulation. Note the profound disruption in the EPSP post-injury in area CA1 and in the dentate when stimulating the mPP (arrows). A distinct pattern commonly appears in the waveforms post-injury that may be due to the population activity response invading the EPSP, suggesting hyper-excitability in the neuronal response.

FIG. 3.

Changes in paired pulse facilitation and depression post-TBI. (A) Paired pulse facilitation (PPF) as demonstrated in area CA1, where an increased slope (S2) was evident in the second pulse (50 msec interpulse interval). An analysis of variance including injury group and an interaction with brain region yielded evidence of a significant main effect of injury [F(1,6) = 11.5, p = 0.015], as well as evidence of differences in the effect of injury in the three different brain regions [F(2,98) = 3.29, p = 0.042]. (B) Compared with sham, recordings in area CA1 suggested PPF was reduced by a factor of 0.79 (95% CI 0.69, 0.91; p = 0.006] following mild head rotation. (C) Responses to stimulation in the dentate (medial perforant path [mPP]) suggested injured animals showed a reduction of 0.91-fold (95% CI 0.80, 1.05) relative to sham animals (p = 0.18). (D) Similar PPF occurred in sham recordings in the lateral perforant path (lPP), with a reduction of 0.90-fold (95% CI 0.78, 1.03) in injured versus sham animals (p = 0.097). Note that these results for mPP and lPP did not achieve statistical significance. To visualize both between animal variation as well as within-animal variation arising from the multiple slices, the data are presented as boxplots for each individual animal; the horizontal line is the median, the box is the interquartile range (IQR) and each vertical line is the box ±1.5 IQR truncated at the minimum and maximum values for each animal. Outliers are shown as individual points.

FIG. 4.

Decrease in fiber volleys in CA1 at 7 days following mTBI. On average fiber volley slope in area CA1 was a factor of 0.52-fold (95% CI 0.47, 0.93) lower post-injury than sham (F(1,6) 0 = 8.3, p < 0.028), with no evidence of an injury interaction with current [F(9, 421) = 0.78, p = 0.63]. In region medial perforant path (mPP), the mean fiber volley slope of injured animals was a factor of 0.90-fold (95% CI 0.66, 1.313), compared with sham, [F(1,6) = 0.27, p = 0.62, NS for the main effect and F(9, 432) = 0.38, p = 0.95, NS for the interaction]. In region lPP fiber volley slope was a factor of 0.66-fold (95% CI 0.58, 1.19) lower post-injury than sham [F(1,6) = 4.67, p = 0.25 and F(9, 432) = 1.72, p = 0.083 for the interaction] but also not significant. Data shown are empirical means ± standard error of the mean (SEM) where the SEM is based on the number of animals per group.

FIG. 5.

Post-TBI output (EPSP) levels as a function of input (FV slope). (A) Input/output curves were generated via stimulation of the Schaffer collaterals and medial and lateral perforant path in the dentate region. The injured group trended towards higher mean EPSP levels in CA1 and medial perforant path, and lower levels in lateral perforant path, but these were not significant at individual current levels. (B) The empirical mean EPSP is plotted as a function of mean FV slope for each level of current. In the CA1 region we found strong evidence of an interaction between injury and FV slope [F(2, 428) = 6.95, p = 0.001], indicating the presence of an injury effect that differed as a function of FV slope. These results suggest that for a given input (FV) in area CA1, there is a greater output (EPSP) post injury. The results for this interaction for medial perforant path (mPP) did not achieve statistical significance [F(2, 437) = 2.28, p = 0.10] and there was no evidence of an effect of injury by FV slope interaction in the lateral perforant path (lPP) [F(2,447) = 0.35, p = 0.70, NS]. There was also no evidence of an overall effect of injury in the lPP [F(1,6) = 0.023, p = 0.89 NS]. Data presented as animal mean ± standard error of the mean.

FIG. 6.

Lack of axonal pathology in the hippocampus despite functional alterations. At 7 days after injury or sham conditions, axonal degeneration was assessed both within hippocampal pathways, as well outside the hippocampal structure in sub-cortical white matter tracts (as an internal positive control). Representative micrographs from sham animals are presented in the left column, and from injured animals in the right column. (A, B) In sub-cortical white matter, amyloid precursor protein (APP) immunoreactive axons displayed the classic morphological appearance of traumatic axonal injury, including terminally disconnected swollen axonal bulbs, following TBI (A) but were absent in sham animals (B). However, no overt APP accumulation was observed within the hippocampal formation in any animals following injury, including regions that corresponded to the stimulation/recording sites on the contralateral side (i.e., the stratum moleculare layer of the dentate gyrus and the stratum radiatum of the CA1 region) as demonstrated in representative hippocampal sub-fields following sham conditions (C, E) or head rotation (D, F). Moreover, neurofilament-200 immunolabeling revealed axons of normal density, morphology and distribution in these regions, with consistent appearance between sham (G, I) and injured (H, J) animals. Collectively, these observations suggest the functional compromise of intra-hippocampal axons may not be explained by overt axonal loss/degeneration or transport disruption. Scale bar = 25 μM for (A) and (B), 50 μM for (C-J).