Long-term cognitive deficits after traumatic brain injury associated with microglia activation

Highlights

• Traumatic brain injury induces long-term cognitive deficits in mice.

• Injury induces the activation of microglia and CNS infiltration by macrophages.

• After injury, microglia change phenotypes and the frequency of activated microglia increases with the injury intensity.

• Activated microglia found in injured brains correlate positively with memory deficit 35 days post injury.

Abstract

Traumatic Brain Injury (TBI) is the most prevalent of all head injuries. Microglia play an essential role in homeostasis and diseases of the central nervous system. We hypothesize that microglia may play a beneficial or detrimental role in TBI depending on their state of activation and duration.

In this study, we evaluated whether TBI results in a spatiotemporal change in microglia phenotype and whether it affects sensory-motor or learning and memory functions in male C57BL/6 mice. We used a panel of neurological and behavioral tests and a multi-color flow cytometry-based data analysis followed by unsupervised clustering to evaluate isolated microglia from injured brain tissue. We characterized several microglial phenotypes and their association with cognitive deficits. TBI results in a spatiotemporal increase in activated microglia that correlated negatively with spatial learning and memory at 35 days post-injury. These observations could define therapeutic windows and accelerate translational research to improve patient outcomes.

Introduction

Microglia are the central nervous system (CNS) resident innate immune cells that play critical physiological roles in the healthy and injured brain. They detect and rapidly respond to any disruption in the status quo of the CNS, including infections or tissue injury, and often act to remove cellular debris [1]. The activation of microglia from their resting surveillance state occurs within minutes of the injury and is critical for recovery. However, prolonged activation may be detrimental and contribute to secondary damage. When the microglia are activated in response to any disruption, this activation alters their gene expression and morphology [2]. Although they have distinctive lineage, microglia resemble blood-derived macrophages that infiltrate the CNS from the periphery in response to tissue damage [3]. Microglia are usually classified into the classical M1 phenotype that is considered proinflammatory, and the M2 anti-inflammatory is thought to be involved in neural repair [4]. However, more recent data suggest that microglia's activation status is on a continuum between anti- and pro-inflammation rather than a rigid dichotomous phenotype [5].

Chronic neuroinflammatory response to an acquired brain insult such as a TBI contributes to the injury and lengthens or halts recovery [6]. In chronic neuroinflammation, microglia remain activated for an extended period during which the production of repair mediators is sustained longer than usual [7]. Of note, chronic microglial activation has also been linked to most, if not all, neurodegenerative diseases like Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease [[8], [9], [10]]. In humans with traumatic brain injury (TBI), microglial activation has been reported as early as 72 h after injury [11], and it can persist for months after injury [12].

TBI is the most prevalent of all CNS injuries and results in chronic neurologic and cognitive deficits [13]. TBI causes cell death and neurologic dysfunction through secondary injury mechanisms characterized by edema, neuronal cell death, glial activation, and infiltration of peripheral immune cells [14]. Extensive research has been conducted investigating the role of microglia after head injury and its interaction with the neural microenvironment suggesting a role for microglia in the injury process as well as in neurotransmission and maintenance of synaptic integrity [15]. TBI initiates a neuroinflammatory cascade that persists over time and may lead to prolonged cognitive deficits. The regional distribution of microglial activation is yet to be determined. Some studies found chronic activation in the thalamus, others in the hippocampus in addition to the injury site [16]. Despite evidence showing a significant role for microglia in the pathogenesis of TBI, no studies to date have examined the spatiotemporal microglial activation response after injury and explored its potential correlation with cognitive deficits [[17], [18], [19]].

Here we hypothesized that TBI would result in phenotypic changes in the microglial cell population that persists for a long time after the injury. We also hypothesized that chronic microglial activation is associated with cognitive deficits. We analyzed the spatiotemporal course of microglia changes isolated from the injured brain up to 35 days after controlled cortical impact (CCI) in mice. We used conventional flow cytometry techniques followed by bioinformatics-based multi-parametric methods that are not constrained by assumptions or bias. We observed heterogeneity in microglia phenotypes and temporal changes in microglia subpopulations following TBI. A particular subpopulation that we called hyperactivated microglia was correlated with chronic deficits in learning and spatial memory after injury.