Elsevier

Experimental Neurology

Volume 275, Part 3, January 2016, Pages 427-435
Experimental Neurology

Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury

https://doi.org/10.1016/j.expneurol.2015.07.016Get rights and content

Highlights

  • Reduced mean diffusivity and radial diffusivity was observed in cortex region at day 3 and day 5 post injury in rodent model of mild TBI.

  • Significant correlation was observed between increased GFAP expression and DTI indices during acute phase of mTBI.

  • This study suggest that MD and RD are potential markers of injury and may help in assessing/monitoring of mTBI during acute phase of injury.

  • Present findings corroborate with deficits and pathologies in clinical mTBI, and support the model to address pathology and therapeutic options.

Abstract

The majority of human mild traumatic brain injuries (mTBI; 70%) lack radiological evidence of injury, yet may present long term cognitive, and behavioral dysfunctions. With the hypothesis of evident damaged neural tissue and immunological consequences during acute phase of mTBI, we used closed skull weight-drop TBI model to address human mTBI condition. Serum cytokines (TNF-α, IL-10) and glial fibrillary acidic protein (GFAP) expression were examined at day 0 (control, pre-injury), 4 h, day 1, day 3 and day 5 post injury (PI). Diffusion tensor imaging (DTI) was performed at similar timepoints to identify neuroinflammation translation into imaging abnormalities and monitor injury progression. DTI indices including mean diffusivity (MD), radial diffusivity (RD), fractional anisotropy and axial diffusivity values were quantified from cortex (CTX), hippocampus and corpus callosum regions. One way ANOVA showed significant increase in TNF-α at 4 h and IL-10 at day 1 PI as compared to control. GFAP+ cells were significantly increased at day 3 and day 5 as compared to control in CTX. Repeated measures ANOVA revealed significant decreases in MD, RD values in CTX at day 3 and day 5 as compared to day 0. A significant, inverse correlation was observed between cortical MD (r =  0.74, p = 0.01), AD (r =  0.60, p = 0.03) and RD (r =  0.72, p = 0.01) values with mean GFAP+ cells in the cortical region. These findings suggest that mTBI leads to elevated cytokine expression and subsequent hypertrophy of astrocytic processes. The increased numbers of reactive glial cells contribute diffusion restrictions in the CNS leading to reduced MD and RD values. These findings are in line with the deficits and pathologies associated with clinical mTBI, and support the use of mTBI model to address pathology and therapeutic options.

Introduction

Mild traumatic brain injury (mTBI) has been reported as the most prevalent form (70%) of all the head injuries (Arciniegas et al., 2005, Jennett, 1998). Due to the absence of immediate disabling symptoms at the time of injury and less often overtly visible consequences, mTBI is also referred as the “silent epidemic” (National Center for Injury Prevention and Control Report, 2003). Human studies on mTBI have shown persistent neurocognitive deficits in approximately 15% of patients (Roe et al., 2009, Hartlage et al., 2001) with abnormalities in cerebral blood flow, (Ge et al., 2009, Jacobs et al., 1996) symptoms of post-concussive syndrome, (Bazarian et al., 1999, Masson et al., 1996, Iverson et al., 2006) and deficits in cognitive and executive function (Bemstein, 2002, Kraus et al., 2007) for months to years after mTBI. Additionally, preliminary studies show evidence of cognitive deficits in mTBI associated with specific functional or neuroanatomical lesions (Jacobs et al., 1996, Chen et al., 2004).

In view of the prevalence and outcome of mTBI, various experimental studies have been conducted. In vivo studies have failed to show any apparent cell death and tissue damage in mTBI that is commonly found after moderate and severe TBI (Dixon et al., 1991, Scheff et al., 1997, DeFord et al., 2002). Animal models of non-penetrative closed head mTBI have demonstrated resultant cellular dysfunction (Lyeth et al., 1990, Kanayama et al., 1996, Uryu et al., 2002), cognitive and behavioral short- and long-term deficits (Edut et al., 2011, Milman et al., 2005, Rachmany et al., 2013, Tweedie et al., 2007, Tweedie et al., 2013, Zohar et al., 2003, Marklund and Hillered, 2011).

The course of injury involves primary and secondary brain injury which occurs immediately after insult to the head and minutes to days following trauma respectively (Greve and Zink, 2009). These secondary events of the trauma may lead to edema in the brain and inflammatory consequences (Choi et al., 1987, Cornelius et al., 2013, Maas et al., 2008). There is quick release and rapid sequestration of various pro- and anti-inflammatory cytokines into the CNS and blood stream in response to TBI (Lu et al., 2009). Experimental TBI models show enhanced TNF-α and IL-1β peaks within 3–8 h after injury, followed by sustained elevations of IL-6 and IL-10 (Kirchhoff et al., 2008). During the acute phase of injury, TNF-α and IL-10 are produced in high concentrations by resident microglia and infiltrating monocytes/macrophages (D'Mello et al., 2009). Subsequently, these elevated inflammatory cytokines stimulate astrocyte reactivity leading to a cascade of increased neuroinflammation and development of secondary injury following neurotrauma (McKeating and Andrews, 1998). Studies on severe TBI patients have shown that glial fibrillary acidic protein (GFAP) concentrations were associated with injury severity and outcome (Vos et al., 2010, Papa et al., 2012). It has also been shown to be predictive of elevated intracranial pressure, reduced mean arterial pressure, low cerebral perfusion pressure, poor Glasgow Outcome Score (GOS) and increased mortality (Pelinka et al., 2004, Honda et al., 2010). GFAP is a highly brain-specific monomeric intermediate filament protein that is almost exclusively expressed in astroglia (Olsson et al., 2011). A recent study of 108 patients with mild or moderate TBI, demonstrated elevated levels of GFAP in the serum within 1 h after injury, discerning TBI patients from uninjured controls (Honda et al., 2010). The same study also reported a significant difference in GFAP levels between mild TBI (GCS 15) and general trauma controls (Papa et al., 2012).

mTBI has been defined as a condition of normal structural imaging by World Health Organization (WHO) guidelines (Cassidy et al., 2004) because often conventional computed tomography (CT) and magnetic resonance imaging (MRI) techniques are not sensitive in detecting diffuse/traumatic axonal injuries, which constitute the majority of brain injuries observed in mTBI (Benson et al., 2007). This lack of radiological evidence of brain injury in mTBI has led to the development of more sensitive methods like diffusion tensor imaging (DTI) to assess subtle alterations in brain morphology that may underlie mTBI (Belanger et al., 2007). DTI measures the directional diffusion of water present in the tissue that varies depending on tissue type and pathology (Le Bihan, 1991). In normal brain tissue, physical boundaries of white matter restrict the diffusion of water, favoring movement of water parallel and restricting it perpendicular to the axons. Microstructural axonal injuries could be revealed using DTI (Basser et al., 1994, Pierpaoli and Basser, 1996) which are believed to be potentially responsible for symptoms following mTBI. mTBI alone is considered to be non-fatal thereby leaving scant evidence of neuropathological studies (Browne et al., 2011).

Available literature on experimental models of TBI indicate injury induction via fluid percussion or controlled cortical impact, where skull is invariably removed and direct trauma to the brain tissue, produces moderate to severe TBI (Morales et al., 2005) resulting in focal lesions, blood–brain barrier (BBB) disturbances, edema formation, and morphologically evident brain damage (Carbonell and Grady, 1999, Graham et al., 2000, Thompson et al., 2005). Since craniotomy is indispensable in these models, mTBI models developed through these methods may lead to ignoring some pathophysiological alterations speculated in mTBI. Therefore a clinically relevant closed head injury model is being investigated in the present study. Similar model has been studied earlier (De Mulder et al., 2000, Engelborghs et al., 1998, Engelborghs et al., 2000; Rooker et al., 2002) and characterized by several clinically relevant features, including increased ICP, diffuse axonal injury, contusions, impairment of cerebral blood flow autoregulation, and reduction of brain oxygenation. A modified weight drop method for adult rats and mice of mild closed head injury that more closely resembles a human concussion injury has also been developed (Henninger et al., 2005, Henninger et al., 2007, Tang et al., 1997). These experimental models do not lead to gross morphological damage, but discrete cell loss in the rat cortex and hippocampus 9–14 days PI, accompanied by deficits in spatial learning and memory (Fujiki et al., 2008, Henninger et al., 2005, Tang et al., 1997). None of these studies measured acute inflammatory markers and GFAP levels which are an indicator of injury severity and outcome. They however showed normal β-APP and MAP-2 levels with neuronal and histopathological alterations. Using a modified model of mTBI (with normal conventional MRI) in young adult male rats, we examined if this injury resulted in early inflammatory response, escalated GFAP levels (which are an indicator of injury and have been shown in human TBI) and abnormality on DTI indices (indicating translation of injury and providing a non-invasive method of monitoring trauma pathology).

Owing to neuropathological alterations and neuropsychological deficits in presence of normal conventional imaging in mTBI patients, the present study aims to assess altered glial/inflammatory measures with DTI findings in acute phase of mild TBI.

Section snippets

Methods

Forty-five adult, male Sprague–Dawley rats (8–10 weeks, 200–250 g), housed (23 °C ± 1 °C, 50% ± 5% humidity, and 12 h light/dark cycle) with free access to food and water were used during the study. Out of the 45 rats, 10 were used only for MR imaging till day 5. The remaining 35 rats were randomly assigned into 5 groups of 7 rats each, for inflammatory cytokines and GFAP analysis at 4 h, 1 day, 3 days, and 5 days post injury (PI) and control group (0 day). Out of these 35 animals, 15 animals underwent both

Imaging findings

We found none of the animals showed any contusion injury/tissue loss in the cerebral hemispheres on turbo RARE images at any timepoint after injury. Mean values ± SD of various DTI indices from all ROI's on rat brain parenchyma for all timepoints are listed in Table 1.

In vivo temporal alterations in diffusivity indices during acute phase of mTBI

Injury induced decreased MD values were observed in the cortical gray matter region (nearest to the site of impact) as compared to controls at all timepoints PI which reached the level of statistical significance at day 3 and day 5 (

Discussion

In the present study, we have tried to investigate the neuroimflammatory and accompanying microstructural alterations in acute phase of closed head, weight-drop rodent model of mTBI, when no injury was visible on conventional MRI. The cascade of injury induced events may include blood brain barrier (BBB) breakdown, edema formation, swelling with subsequent infiltration of blood cells leading to elevated levels of inflammatory cytokines and activation of resident glial cells (Perez-Polo et al.,

Conflict of interest

Authors have no conflict of interest to declare.

Acknowledgments

Ms. Kavita Singh would like to acknowledge the fellowship grant from Department of Science and Technology (DST), New Delhi, India. We acknowledge Dr. I Namita for her assistance with histological study.

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