Elsevier

Experimental Neurology

Volume 213, Issue 2, October 2008, Pages 372-380
Experimental Neurology

Exacerbated glial response in the aged mouse hippocampus following controlled cortical impact injury

https://doi.org/10.1016/j.expneurol.2008.06.013Get rights and content

Abstract

Old age is associated with enhanced susceptibility to and poor recovery from brain injury. An exacerbated microglial and astrocyte response to brain injury might be involved in poor outcomes observed in the elderly. The present study was therefore designed to quantitate the expression of markers of microglia and astrocyte activation using real-time RT-PCR, immunoblot and immunohistochemical analysis in aging brain in response to brain injury. We examined the hippocampus, a region that undergoes secondary neuron death, in aged (21–24 months) and adult (5–6 months) mice following controlled cortical impact (CCI) injury to the sensorimotor cortex. Basal mRNA expression of CD11b and Iba1, markers of activated microglia, was higher in aged hippocampus as compared to the adult. The mRNA expression of microglial markers increased and reached maximum 3 days post-injury in both adult and aged mice, but was higher in the aged mice at all time points studied, and in the aged mice the return to baseline levels was delayed. Basal mRNA expression of GFAP and S100B, markers of activated astrocytes, was higher in aged mice. Both markers increased and reached maximum 7 days post-injury. The mRNA expression of astrocyte markers returned to near basal levels rapidly after injury in the adult mice, whereas again in the aged mice return to baseline was delayed. Immunochemical analysis using Iba1 and GFAP antibodies indicated accentuated glial responses in the aged hippocampus after injury. The pronounced and prolonged activation of microglia and astrocytes in hippocampus may contribute to worse cognitive outcomes in the elderly following TBI.

Introduction

In the United States, an estimated 1.5 million people sustain traumatic brain injury (TBI) every year; about one every 21 s (Letarte, 2006). The cumulative burden of this epidemic is estimated to be 5.3 million Americans, which is nearly 2% of the population, living with disabilities resulting from TBI. The Centers for Disease Control and Prevention analyzed TBI-related hospitalizations by age and found that TBI most commonly occurs in adolescents and young adults aged 15 to 24 years, and in the elderly (75 years and older). The incidence rates of TBI, starting at age 65, have been observed to double for every additional 10 years of age (Coronado et al., 2005). Persons aged ≥ 75 years had the highest TBI-related hospitalization rate, at least twice the rate of any other age group (Coronado et al., 2005). Older age is a variable known to negatively influence outcome after TBI (Hukkelhoven et al., 2003). Studies indicate that outcome and mortality rates are generally worse for older people than for younger people with similar injuries (Ferrell and Tanev, 2002, Mohindra, et al., in press; Rapoport et al., 2006). The aging of the United States population will further increase the incidence and associated disability of non-fatal TBI (Brown et al., 2004).

Long-term cognitive and motor dysfunctions have been recognized as the most debilitating consequences of human TBI (Klein et al., 1996). TBI results in neurological dysfunction through primary and secondary (delayed) brain injury. Primary injury occurs at the time of the insult, whereas secondary injury occurs over a period of days. Secondary brain injury is incurred as a result of pathological processes initiated at the time of the primary injury and may be amenable to treatment, thereby improving prognosis. Secondary molecular, biochemical, and cellular events cause neuronal injury and loss in multiple brain areas, including the hippocampus (Bigler et al., 2002). In both humans and rodents, age exacerbates the cognitive decline following TBI, suggesting synergistic action (Goldstein and Levin, 2001, Johnstone et al., 1998, White-Gbadebo and Hamm, 1993). In addition, the hippocampus has been demonstrated to be a selectively vulnerable region after experimental TBI with neurons undergoing apoptotic death (Rink et al., 1995).

We have recently investigated the effect of age on outcomes after controlled cortical impact (CCI) injury in the mouse using behavioral, magnetic resonance imaging and histological studies and found higher vulnerability of the aged brain to neurodegeneration along with diminished functional recovery (Onyszchuk et al., 2008). Furthermore, more dying neurons were observed in hippocampus of aged mice following CCI. The mechanism of worse outcome in aging is unknown, but aging has been shown to exaggerate the inflammatory phase of wound healing, a finding that may be causally related to slower wound healing in older subjects (Ashcroft et al., 2002).

Neuroinflammation contributes to the cascade of events that are responsible for development of secondary brain damage and adverse outcome following TBI (Schmidt et al., 2004). In the context of inflammatory mechanisms, glial cells are recognized to play an active role in most degenerative CNS pathologies, and reactive gliosis is recognized as a universal hallmark of both acute or chronic damage to the CNS (Marchetti and Abbracchio, 2005). Glial activation in response to cytokine exposure is enhanced in the aged brain (Deng et al., 2006, Godbout et al., 2005), and previous work from our laboratory has demonstrated higher expression of pro-inflammatory cytokines and chemokines during secondary neuron death in thalamus of aged mice following cortical aspiration injury (Sandhir et al., 2004).

Despite extensive evidence implicating inflammatory mechanisms in CNS damage following traumatic brain injury, the influence of age on microglial and astrocytic responses has not been investigated. This study was therefore designed to quantitate glial responses in the hippocampus of aged (21–24 mo) and adult (5–6 mo) mice after CCI injury to the sensorimotor cortex by examining the expression of markers of microglia and astrocyte activation using real-time RT-PCR, western blot and immunochemical analysis.

Section snippets

Animals

Studies were performed using adult (28–32 g, 5–6 months old) and aged (28–32 g, 21–24 months old) C57BL/6 male mice obtained from the National Institute of Aging colonies, which were barrier raised, monitored for genetic purity and screened for bacterial and viral pathogens strictly according to the NIA guidelines. The animals were housed in the AAALAC-accredited Laboratory Animal Resources (LAR) of the University of Kansas Medical Center and acclimatized for 2 weeks before commencing

Microglial activation in hippocampus following CCI Injury

Hippocampal mRNA levels of microglial activation markers, CD11b and Iba1, were determined using real-time RT-PCR 1, 2, 3, 7, 14 and 28 days following CCI injury in hippocampus of adult and aged mice (Fig. 2). Basal levels of both CD11b and Iba1 mRNA were higher in the aged hippocampus than in the adult tissue. Following injury, expression of CD11b and Iba1 increased after 24 h and peaked at 3 days in both the adult and aged mice. Both microglial markers were significantly higher in the aged

Discussion

Neuroinflammation following acute brain trauma plays a prominent role in both pathological and reconstructive response of the brain to injury (Lucas et al., 2006). Activated microglia and astrocytes play a crucial role in the neuroinflammatory process. Activated glial cells contribute to the pathogenesis and progression of neurological disorders ranging from traumatic brain injury to Alzheimer's disease (Maeda et al., 2007). While the glial response can provide trophic and metabolic support to

Acknowledgments

The authors acknowledge the assistance of Eugene Gregory in carrying out the work and Eileen Roach with processing of the images. Special thanks to Dr. Y.Y. He for his help with controlled cortical impact injury. The study was supported in part by the Steve Palermo Endowment and grant from the National Institute of Aging (AG026482 and P30 NICHD HD 02528).

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