Complement factor C1q mediates sleep spindle loss and epileptic spikes after mild brain injury

Abstract

Although traumatic brain injury (TBI) acutely disrupts the cortex, most TBI-related disabilities reflect secondary injuries that accrue over time. The thalamus is a likely site of secondary damage because of its reciprocal connections with the cortex. Using a mouse model of mild TBI (mTBI), we found a chronic increase in C1q expression specifically in the corticothalamic system. Increased C1q expression colocalized with neuron loss and chronic inflammation and correlated with disruption in sleep spindles and emergence of epileptic activities. Blocking C1q counteracted these outcomes, suggesting that C1q is a disease modifier in mTBI. Single-nucleus RNA sequencing demonstrated that microglia are a source of thalamic C1q. The corticothalamic circuit could thus be a new target for treating TBI-related disabilities.

Fig. 1.. The injured cortex and functionally connected thalamus show chronic inflammation and neuron loss three weeks after mTBI.

(A, B) Schematic of the controlled cortical impact (A) and of the S1 cortex and nRT and VB thalamic regions (B). (C) Coronal brain section from a mTBI mouse stained for C1q. Note that bilateral C1q expression is typical for the hippocampus. (D) Close-up images of S1 (top), VB and nRT (middle), and confocal images of nRT (bottom) stained for C1q, NeuN, GFAP, and Iba1. Injury site in the right S1 cortex is marked by an asterisk. Arrow in nRT indicates location of confocal image. (E) Fluorescence ratios between ipsilateral and contralateral regions in sham and mTBI mice. Data represent all points from min to max, with a Mann-Whitney test and α = 0.05 (*p < 0.05, **p < 0.01). Analysis includes between five and seven mice per group (n = three sections per mouse, one image per region).

Fig. 2.. The nRT ipsilateral to the injured cortex shows neuron loss and altered sIPSC and sEPSC properties three weeks after mTBI.

(A-C) High-magnification coronal image of the nRT (A) and neuron counts across the entire ipsilateral nRT (B) or per subdivision, normalized to the median value from the sham group (C). Six mice per group (n = three sections per mouse, averaged). (D, E) sIPSC recordings from nRT (D) neurons and frequency and amplitude distributions (E) in 13 neurons from four sham mice and 22 neurons from six mTBI mice. (F, G) Spontaneous EPSC recordings (F) from nRT neurons and frequency and amplitude distributions (G) in 11 neurons from six sham mice and nine neurons from seven mTBI mice. Inset: averaged EPSC traces from single nRT neurons. (H) Coronal brain sections from Thy1-GCaMP6f mice with sham surgery and mTBI (asterisk). Bottom: projection terminals from the cortex. (I) Thy1-GCaMP fluorescence ratios between ipsilateral and contralateral regions. Data represent all points from min to max, Mean ± SEM, with a Mann-Whitney test and α = 0.05 (*p < 0.05, **p < 0.01). Analysis includes five sham mice and six mTBI mice (n = three sections per mouse, one image per region).

Fig. 3.. Microglia are a source of C1q in the thalamus three weeks after mTBI.

(A) Schematic of coronal brain sections showing the location of thalamic tissue dissection. (B, C, E) UMAP representation of single-nucleus RNA sequencing data (n = 4,908 sham nuclei, n = 4,338 mTBI nuclei, after data cleaning) colored by cell type or lineage (B), and for nuclear C1qa (C) or C4b (E) expression. Lineage markers described in Fig. S2A. Normalized expression scale shown above, 0-max, with max value for each panel. (D, F) Violin plots of C1qa expression in microglial nuclei (D) and of C4b expression in oligodendrocyte nuclei (F) from cluster 3 (Oligo 3, Fig. S3E) from sham and mTBI mice, analyzed with a Wilcoxon Rank Sum test (ns = not significant). Analysis combines both technical replicates, collectively representing nine sham mice and ten mTBI mice. Each dot represents a single nucleus.

Fig. 4.. Anti-C1q antibody reduces chronic inflammation and neuron loss three weeks after mTBI.

(A, B) Coronal brain sections (A) and close-ups (B) of S1 (top), VB and nRT (bottom) from mTBI mice treated with anti-C1q antibody and stained for C1q, NeuN, GFAP, and Iba1. Asterisk marks injury site. (C) Quantification of nRT neuron counts and fluorescence ratios between ipsilateral and contralateral regions in sham and mTBI mice treated with the anti-C1q antibody or the isotype control. Data represent all points from min to max, with a Mann-Whitney test and α = 0.05 (*p < 0.05, **p < 0.01). Analysis includes between six and eight mice per group (n = three sections per mouse, one image per region).

Fig. 5.. Anti-C1q antibody reverts chronic sleep spindle reduction after mTBI.

(A) ECoG recordings from a sham and mTBI mouse three weeks after mTBI. Blue traces represent the band-pass (BP, 8—15 Hz) filtered ECoG. Horizontal blue lines show the detected spindles. Arrows indicate epileptic spikes. (B) Same as (A) from mTBI mice treated with an isotype control or the anti-C1q antibody. (C) Ratio of sleep spindle counts in ipsilateral ECoG to sleep spindle counts in contralateral ECoG detected within a 12-hour window. Data represent mean ± SEM analyzed with a Mann-Whitney rank sum test with α = 0.05 (*p < 0.05, **p < 0.01). Analysis includes n = six sham mice, n = nine mTBI mice (left); n = seven control-treated mTBI mice, n = six antibody-treated mTBI mice (right). (D) Density, normalized amplitude and duration of sleep spindles in contralateral and ipsilateral ECoG from the mice in (C). Data represent mean ± SEM analyzed with a Kruskal-Wallis One Way Analysis of Variance on Ranks, all pairwise multiple comparison procedures (Holm-Sidak method), α = 0.05 (*p < 0.05, **p < 0.01). Gray lines connect contralateral and ipsilateral data points for each mouse.

Fig. 6.. Anti-C1q antibody reduces focal epileptic spikes that develop three weeks after mTBI.

(A) ECoG recordings from a sham and mTBI mouse three weeks post-mTBI. Horizontal dashed lines represent the spike detection threshold. Vertical red lines indicate detected spikes. (B) Same as A) from mTBI mice treated with an isotype control or the anti-C1q antibody. Traces in A-B are from episodes of NREM sleep. (C) Number of epileptic spikes detected within a 12-hour window. Data represent mean ± SEM analyzed with a Mann-Whitney rank sum test, α = 0.05 (*p < 0.05, **p < 0.01). Inset: an average epileptic spike from the mTBI mouse shown in (B) (n=592 spikes; mean (black) ± SD (grey). Analysis includes n = six sham mice, n = nine mTBI mice (left); n = seven control-treated mTBI mice, n = six antibody-treated mTBI mice. (D, E) Number of epileptic spikes as a function of the ratio of sleep spindles in the sham versus mTBI (D) and anti-C1q versus control mTBI (E) mice from (C). Individual points represent each mouse and error bars represent mean ± SEM across both axes.