Elsevier

Experimental Neurology

Volume 316, June 2019, Pages 39-51
Experimental Neurology

Research paper
Depletion of microglia immediately following traumatic brain injury in the pediatric rat: Implications for cellular and behavioral pathology

https://doi.org/10.1016/j.expneurol.2019.04.004Get rights and content

Abstract

The inflammatory response is a significant component of the pathophysiology of pediatric traumatic brain injury. High levels of inflammatory mediators have been found in the cerebrospinal fluid of brain-injured children which have been linked to poor prognosis. Targeting aspects of the inflammatory response in the hopes of finding a viable post-injury therapeutic option has gained attention. Microglia are largely responsible for perpetuating the injury-induced inflammatory response but in the developing brain they play beneficial roles in both normal and disease states. Following closed head injury in the neonate rat, depletion of microglia with intracerebral injections of liposomes containing clodronate was associated with an increase in neurodegeneration in the early post-injury period (3 days) relative to those injected with empty liposomes suggestive of a decrease in clearance of dying cells. In sham-injured animals, microglia repopulated the clodrosome-mediated depleted brain regions over a period of 2–4 weeks and exhibited morphology typical of a resting phenotype. In brain-injured animals, the repopulated microglia in clodrosome-injected animals exhibited rod-like and amoeboid morphologies. However, fluoro-Jade B reactivity in these brain regions was more extensive than in empty liposome-injected animals suggesting that the active microglia may be unable to clear dying neurons. This was accompanied by an induction of hyperexcitability in the local cortical circuitry. Depletion of microglia within the white matter tracts and the thalamus did not affect the extent of injury-induced traumatic axonal injury. Increased neurodegeneration in the dorsal subiculum was not accompanied by any changes to injury-induced deficits in spatial learning and memory. These data suggest that activation of microglia may be important for removal of dying neurons in the traumatically-injured immature brain.

Introduction

Traumatic brain injury (TBI) remains one of the leading causes of morbidity and mortality in infants and children <4 years or age (Anderson et al., 2005; Coronado et al., 2011; Emami et al., 2017; Faul et al., 2010; Langlois et al., 2005). Improvements in supportive care in the acute post-traumatic period over the past decade has resulted in decreased mortality (C.D.C., 2016) although survivors are faced with a lifetime of behavioral deficits. Among the many complications that manifest over the lifespan of survivors of infant TBI are impairments of learning and memory, language and executive function all of which are often associated with pathologic alterations in both white and gray matter (Anderson et al., 2009, Anderson et al., 2011; Babikian et al., 2015; Catroppa and Anderson, 2004, Catroppa and Anderson, 2006; Dennis et al., 2015; Ewing-Cobbs et al., 2006; Ewing-Cobbs et al., 2004; Power et al., 2007; Salorio et al., 2005; Tong et al., 2004; Wilde et al., 2005). The mechanisms underlying these pathologic alterations are incompletely understood although activation of microglia leading to the synthesis and release of cytokines and chemokines, may play an important role (Loane and Kumar, 2016; Woodcock and Morganti-Kossmann, 2013). Following severe TBI in children, increase in pro- and anti-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and IL-10, chemokines IL-8 and C—C motif ligand-3, and nucleotide-binding domain-like receptor protein-mediated inflammasome, and quinolinic acid, ferritin and soluble cluster of differentiation 163 (sCD163), indicative of microglia/macrophage activation in the cerebrospinal fluid has been observed (Bell et al., 1997; Berger et al., 2004; Buttram et al., 2007; Newell et al., 2015; Wallisch et al., 2017; Whalen et al., 2000).

Activation of microglia, the resident immuno-competent cells in the brain, is thought to play an important role in the acute and chronic neurodegeneration observed following brain injury (Beynon and Walker, 2012; Graeber and Streit, 2010; Hanisch and Kettenmann, 2007; Kreutzberg, 1996; Nimmerjahn et al., 2005; Ransohoff and Perry, 2009). The role of microglia in acute and chronic neurodegenerative events following pediatric TBI is being defined. We and others have demonstrated that impact trauma to the immature animal resulted in activation of microglia in multiple brain regions exhibiting evidence of neuronal death and axonal injury and have implicated neuroinflammation as one mechanistic basis for spatial learning and working memory deficits (Chhor et al., 2017; Hanlon et al., 2016; Hanlon et al., 2017; Pullela et al., 2006; Simon et al., 2018; Tong et al., 2002; Zhang et al., 2015). Minocycline, an antibiotic with anti-inflammatory properties, has generally been found to be neuroprotective in multiple animal models of adult or toddler-age TBI (Abdel Baki et al., 2010; Homsi et al., 2009; Homsi et al., 2010; Lam et al., 2013; Sangobowale et al., 2018; Simon et al., 2018; Siopi et al., 2012; Siopi et al., 2011). In contrast, administration of minocycline to the brain-injured neonate rodents reduced microglial proliferation but did not reverse cell death or attenuate spatial learning deficits (Chhor et al., 2017; Hanlon et al., 2016; Hanlon et al., 2017) suggestive of a differential age-at-injury response to treatment.

To evaluate a direct role for microglia activation following pediatric TBI, the present study utilized clodronate (dichloromethylene-bisphosphonate, Cl2MBP) to deplete resident microglia immediately after trauma. Liposome-packaged clodronate is taken up by and induces the apoptosis of phagocytes (Lehenkari et al., 2002; van Rooijen and van Kesteren-Hendrikx, 2003) and has been successful in significantly decreasing microglia numbers in the brain (Asai et al., 2015; Drabek et al., 2012; Faustino et al., 2011; Kumamaru et al., 2012). The hypothesis to be tested in the current study was that by directly removing the resident brain microglia in a clinically-relevant model of pediatric TBI will reduce neuronal and axonal degeneration leading to an attenuation of both functional and behavioral deficits.

Section snippets

Traumatic brain injury

All surgical procedures were done in accordance with the rules and regulations of the Institutional Animal Care and Use Committee at Drexel University College of Medicine. On postnatal day 11, male and female Sprague-Dawley rat pups (Charles River Laboratories, Wilmington MA) were randomly assigned to either receive a closed head injury (N = 53) or treated as sham-injured controls (N = 45) as previously described (Hanlon et al., 2017; Raghupathi and Huh, 2007). Animals were anesthetized using

Acute neurologic outcomes

Closed head injury to the 11-day-old rat resulted in a linear skull fracture below the impact site that was associated with apnea (9–20s) and a loss of righting reflex (Table 1). Brain-injured animals took a significantly longer time to right themselves after injury compared to sham-injured animals after removal of anesthesia (F(3,72) = 22.1, p < 0.00001). Five animals died as a result of the impact (10%); collectively, these data are suggestive of a moderate level of trauma. There were no

Discussion

Depletion of microglia within the site of maximal injury in the cortex led to increased staining for fluoro-Jade B (FJB) in both the acute (3 days) and chronic (35 days) post-injury periods compared to their empty liposome-injected counterparts. This increase in the number of FJB(+) profiles was associated with an increase in the amplitude of the extracellular evoked field potentials suggestive of altered neuronal circuity within the cortex. Depletion of microglia within the white matter tracts

Acknowledgements

The studies were supported, in part, by grants from the National Institutes of Health HD-061963 (RR, JWH) and a fellowship from the Biomedical Studies Graduate Student Association (LAH).

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