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

YEXNR-12080; No. of pages: 9; 4C: Experimental Neurology xxx (2015) xxx–xxx

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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr

Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury☆ Kavita Singh, Richa Trivedi ⁎, M. Memita Devi, Rajendra P. Tripathi, Subash Khushu NMR Research Centre, Institute of Nuclear Medicine and Allied Sciences, Delhi, India

a r t i c l e

i n f o

Article history: Received 29 May 2015 Received in revised form 10 July 2015 Accepted 19 July 2015 Available online xxxx Keywords: Mild traumatic brain injury TNF-α IL-10 Diffusion tensor imaging Glial fibrillary acidic protein Cortex Mean diffusivity Radial diffusivity

a b s t r a c t 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. © 2015 Elsevier Inc. All rights reserved.

1. 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 postconcussive 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

☆ Grant information: This work was performed as a part of Defence Research & Development Organization (DRDO), India sponsored R&D project INM-311. ⁎ Corresponding author at: NMR Research Centre, Institute of Nuclear Medicine and Allied Sciences, Delhi 110054, India. E-mail address: [email protected] (R. Trivedi).

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, 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 proand 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

http://dx.doi.org/10.1016/j.expneurol.2015.07.016 0014-4886/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Singh, K., et al., Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.07.016

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K. Singh et al. / Experimental Neurology xxx (2015) xxx–xxx

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, 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, 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.

2. 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 MRI and GFAP immunostaining for day 0, day 3 and day 5 PI for correlation analysis. All the experimental procedures were approved by institutional ethics committee. Closed head mild injury was induced using modified A. Marmarou's weight drop trauma device (Marmarou et al., 1994). The trauma device consisted of a brass rod of 450 g falling freely by gravity, through an acrylic tube. The brass rod was threaded so that it could be dropped freely from a designated height through a 1-m vertical section of a transparent acrylic tube held in place with an iron stand. The rats were placed on a foam bed embedded in an acrylic box placed on the base of the iron stand. Their heads were immobilized with head holders to limit lateral movements when the head was impacted. A 450 g (1 cm diameter, blunt tip) brass rod was freely dropped from a height of 25 cm above the sagittal midway of the rat brain causing impact at same region and of similar intensity in all the study animals. Foam bed containing the animal was slid to prevent rebound injury after initial impact.

2.1. Imaging protocol Imaging was performed in 10 rats before injury induction at day 0 (each rat served as its own control) and post injury at 4 h, 1 day, 3 days, and 5 days. All MR imaging was performed at a Bruker Biospec 7.0 Tesla (Avance III) 30-cm horizontal bore magnet (Bruker Biospin, Ettlingen, Germany). A four channel rat brain array coil was used as the receiver and a transmit only linear coil was used as the transmitter. Prior to imaging, the rats were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg per kg body weight-BW) and xylazine (10 mg per kg BW). The animal was then placed with head first position on a rat bed and the RF array coil was positioned to cover the entire brain. The animal bed was then slid placed to the center of the magnet bore. A three-slice (axial, mid-sagittal and coronal) scout image using a fast low angle single shot (FLASH) sequence was obtained to localize the rat brain. For localization and identification of anatomical landmarks, a rapid acquisition with relaxation enhancement (RARE) sequence was used to acquire T2 weighted images of the rat brain with parameters of 256 × 256 matrix size, FOV of 4 × 4 cm, repetition time echo time (TR)/(TE) = 2000/13 ms, 1 mm slice thickness with no interslice gap (contiguous slices), and number of slice = 15. DTI images were acquired using a multislice, multiple-shot spin echo EPI sequence with the following parameters: TR/TE = 5000 ms/34.46 ms, number of gradient encoding directions = 46, and b = 672 s mm−2. The other parameters for DTI data acquisition were: acquisition matrix = 128 × 128, field-of-view = 4 cm × 4 cm, slice thickness = 1 mm, and number of slices = 15, number of segments = 4, number of averages = 1, imaging time = 12 min 55 s.

Please cite this article as: Singh, K., et al., Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.07.016

K. Singh et al. / Experimental Neurology xxx (2015) xxx–xxx

2.2. DTI data processing and region of interest analysis The DTI data obtained for different timepoints was processed as described in detail elsewhere (Trivedi et al., 2012). Briefly, after image cropping and distortion corrections, the data was interpolated to attain isotropic voxels and decoded to obtain the tensor field for each voxel. The tensor field data were then diagonalized by using the analytic diagonalization method to obtain the eigenvalues (λ1, λ 2, and λ3) and the three orthonormal eigenvectors. The eigenvalues were used to compute the DTI metrics such as the mean diffusivity (MD), fractional anisotropy (FA), radial diffusivity (RD), axial diffusivity (AD) for each voxel. Regional FA, MD, RD and AD values were obtained by placing regions of interest (ROIs; Fig. 1) on the cortex (CTX), hippocampus (HIPP) and corpus callosum (CC). Bilateral ROIs were placed on all regions except for CC. 2.3. Histological analysis Control rats (day 0) and injury groups (4 h, 1 day, 3 days, 5 days PI) were sacrificed and rat brains were extracted and postfixed in a solution of 10% buffered formaldehyde at 4 °C for 48 h. The intact brains were then blocked and processed in paraffin. Axial (n = 5 brains/group) 6 μm sections were cut (at the level of site of contact during injury) with a microtome (Leica, Germany) and mounted on positively charged glass slides (poly-l-lysine pre-coated). Prior to staining, sections were deparaffinized in xylene, and rehydrated in an ethanol to water gradient. The sections were stained for GFAP (anti-GFAP antibody, 1:100; Sigma). As a negative control, one section was incubated with all reagents except the primary antibody. Tissue sections were subjected to antigen retrieval with heated sodium citrate at 90 °C for 30 min. Endogenous peroxidase activity was quenched with a 15 min H2O2 treatment (0.3%). Each section was rinsed and incubated with the appropriate blocking buffer (ABC staining kit; ImmunoCruz; sc-2017) for 20 min, before applying the appropriate primary antibody overnight at 4 °C. Then, the diluted biotinylated secondary antibody from the ABC staining kit (sc-2017) was applied on each glass slide. Antibodies were detected using the avidin-peroxidase complex, and labeling was revealed after incubating the sections in 3,3′-diaminobenzidine (DAB) peroxidase solution (1% DAB–0.3% H2O2 in 5 ml 0.01 M PBS, pH 7.2) for 3–5 min and counterstained with hematoxylin. 2.4. Microscope analysis and cell quantification Sections were analyzed in the microscope (Dewinter Germany) and immunohistological images were captured for cell counting. Brains images were obtained and quantified for the GFAP+ cells. Multiple field of views (FOV) of each section (4 FOV for CTX bilaterally, 4FOV for HIPP bilaterally and 2 FOV unilaterally for CC) was taken at 10× magnification. The mean of these values was used to calculate the mean number of astrocytes (cells per FOV) in each region. Counting of the cells stained

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positive was performed manually using a computer-based image analysis system (Image J 1.45, Wayne Rasband, National Institutes of Health, Bethesda, USA) by two independent observers. The same protocol was used for all the five groups. GFAP+ cells were counted on histological sections, from five animals, averaged, and calculated as means of counts ± standard deviation per field view for each region/animal. 2.5. Quantitation of serum cytokine levels Blood samples were drawn from the retro orbital plexus of rats before sacrificing them. The samples were kept for 30 min for clot retraction, centrifuged at 5000 rpm for 15 min, and the serum was harvested for TNF-α and IL-10 activity quantitation. Serum TNF-α and IL-10 were measured by enzyme-linked immuno-adsorbent assay (ELISA) using an enzyme-linked immunoassay kit (rat TNF-α ELISA kit, BD OptEIA™, Cat No. 560479 and Rat IL-10 ELISA Set, BD OptEIA™, Cat. No: 555134) according to the manufacturer's protocol. In brief, for TNF-α test, 96well plates containing anti-TNF-α monoclonal antibody were incubated with serum samples and TNF-α standards, diluted with ELISA diluent, for 2 h at room temperature with shaking. A standard curve was established using amounts of TNF-α from 0 to 1600 pg/ml. Plates were incubated at room temperature (RT) for 2 h with anti-rat TNF-α antibody and subsequently washed 5 times. With 30 min incubation at RT with streptavidin-HRP and later 5 washes with buffer, the plates were incubated with ready-to-use TMB substrate for 30 min at room temperature. When the appropriate color developed, the reaction was stopped by adding 100 μl of stop solution to each well and the absorbance was read at 450 nm using a plate reader. TNF-α levels were expressed as pg/ml. For IL-10 activity test, plates were pre coated with 100 μl IL-10 antibody diluted in assay diluent overnight at 4 °C. 100 μl assay diluent was incubated for 1 h after washing 5 times with wash buffer. 100 μl of diluted standards and serum samples were incubated for 1 h followed by working detector incubation of 1 h. Plates were thoroughly washed between steps and ready-to-use TMB substrate was added for appropriate color development for 30 min. The reaction was stopped by adding 50 μl of stop solution to each well and the absorbance was read at 450 nm. IL-10 levels were expressed as ng/ml. 2.6. Statistical analysis A repeated measure ANOVA was performed to assess alterations in diffusion indices with time in different regions of brain. Differences in immunohistological counts of GFAP+ cells, serum TNF-α and IL-10 levels at day 0, 4 h, day 3, and day 5 PI was analyzed using one way ANOVA. Bivariate analysis of correlation with Pearson's correlation coefficient with two-tailed test was performed to study the relationship between the DTI measures in CTX region and GFAP count, with the assumption that there was no correlation between DTI measures and GFAP expression (Ho = 0). Alternatively, if a correlation of b.00 is observed at alpha = .05 and 90% power of the test, the null hypothesis

Fig. 1. Showing images of control rat (day 0). Region of interest (ROI) placement has been depicted over rat axial sections at (A) cortex, (B) hippocampus. ROI placement over (C) ROI for corpus callosum has been shown on RGB-FA map overlaid with MD for better delineation of CC structure where FA threshold of 0.15 is used and white matters structures above threshold are shown in color based on their orientation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Singh, K., et al., Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.07.016

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K. Singh et al. / Experimental Neurology xxx (2015) xxx–xxx

Table 1 showing results of repeated measures ANOVA of DTI indices of AD (axial diffusivity), MD (mean diffusivity), FA (fractional anisotropy) and RD (radial diffusivity) at different timepoints post injury. The values have been indicated as mean ± standard deviation (SD). Regions

Timepoint

MD (×10−3 mm2 s−1) Mean ± SD

Cortex

CC

Hipp

a b

Control 4h 1 day 3 days 5 days Control 4h 1 day 3 days 5 days Control 4h 1 day 3 days 5 days

0.81 ± 0.05 0.79 ± 0.04 0.79 ± 0.05 0.77 ± 0.04 0.78 ± 0.03 0.90 ± 0.07 0.89 ± 0.09 0.87 ± 0.04 0.90 ± 0.06 0.87 ± 0.06 0.83 ± 0.03 0.83 ± 0.03 0.83 ± 0.02 0.83 ± 0.02 0.83 ± 0.02

p-Valuea 0.66 0.14 0.02b 0.04b 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

RD (×10−3 mm2 s−1)

AD (×10−3 mm2 s−1)

FA

Mean ± SD

Mean ± SD

Mean ± SD

0.73 ± 0.05 0.69 ± 0.06 0.70 ± 0.05 0.68 ± 0.05 0.69 ± 0.03 0.58 ± 0.08 0.60 ± 0.11 0.57 ± 0.07 0.60 ± 0.08 0.55 ± 0.08 0.77 ± 0.03 0.77 ± 0.03 0.77 ± 0.02 0.77 ± 0.02 0.77 ± 0.03

p-Value 0.52 0.02b 0.01b 0.02b 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

0.99 ± 0.05 0.98 ± 0.03 0.96 ± 0.06 0.95 ± 0.04 0.95 ± 0.04 1.55 ± 0.11 1.46 ± 0.13 1.49 ± 0.13 1.49 ± 0.16 1.53 ± 0.10 0.95 ± 0.03 0.96 ± 0.03 0.96 ± 0.03 0.96 ± 0.02 0.95 ± 0.02

p-Value 1.00 1.00 0.13 0.13 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

0.22 ± 0.02 0.24 ± 0.06 0.23 ± 0.03 0.24 ± 0.04 0.24 ± 0.03 0.56 ± 0.07 0.51 ± 0.10 0.55 ± 0.08 0.53 ± 0.09 0.58 ± 0.07 0.13 ± 0.01 0.14 ± 0.02 0.14 ± 0.01 0.15 ± 0.02 0.14 ± 0.02

p-Value 0.51 1.00 0.11 0.3 1.00 1.00 1.00 1.00 0.75 1.00 0.79 1.00

p-Values are shown as compared to control. Shows statistically significant values.

Fig. 2. (A) Bar diagram shows changes in DTI measures in the cortex region as a function of time in mTBI model. Significantly decreased mean diffusivity (MD) was observed at day 3 (D3) and day 5 (D5) post injury as compared to control (day 0 — pre injury). Also, radial diffusivity (RD) was found to be significantly decreased at day 1 (D1), day 3 and day 5 post injury. Graph shows mean diffusivities with error bars showing standard deviation. (p-value ≤ 0.05 shown by asterisk marks). (B) Showing (A) control; day 0, (B) day 3 PI; (C) day 5 PI. MD map at 5 day showing decreased MD values (arrow) at site of injury.

Please cite this article as: Singh, K., et al., Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.07.016

K. Singh et al. / Experimental Neurology xxx (2015) xxx–xxx

was rejected. Only p values ≤ 0.05 was considered to be statistically significant (indicated by an asterisk marks). All statistical analysis was done using SPSS 16.0. 3. Results 3.1. 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. 3.2. 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 (Fig. 2). Decrease in MD is evident at day 3 and day 5 in DTI images (Fig. 2-B) as compared to control. MD values were also found to be decreased in the white matter region of corpus callosum at all time point PI but it was non-significant. The other region of hippocampus showed a relatively constant MD values as compared to controls at all the timepoints PI. 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 cortical region (Fig. 3). No significant correlation was observed between FA with GFAP count. RD values, like MD values, showed reductions in cortical gray matter region at all the timepoints as compared to controls which reached the level of statistical significance at day 1, day 3 and day 5. All other regions showed a relatively constant RD values as compared to controls at all timepoints PI. AD, a measure of longitudinal diffusivity, showed non-significant reductions in cortical gray matter as compared to controls at all timepoints PI. All other regions showed relatively stable values of AD at all timepoints PI. FA values showed no significant difference between controls and treated group at any timepoints PI in any region.

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3.3. mTBI increases astrocytes expression Based on the findings of diffusion alterations at day 3 and day 5 PI and evidence of state of astrocyte activation in inflammatory responses post CNS trauma, we used GFAP immunostaining (marker of astrocytes activation) to quantitate the contribution of inflammatory cascade to altered DTI indices in mTBI model. Pronounced upregulation of GFAP expression in astrocytes was observed at day 3 and day 5 PI as compared to controls in the regions of cortical gray matter. Based on the results of independent samples t-test, we found significant increase in GFAP+ cells at day 3 and day 5 in cortical gray matter region as compared to controls (Fig. 4). Other regions of HIPP and CC did not show any significant increase in GFAP+ cells as compared to controls at any timepoint PI. Fig. 5 shows the classical star-like hypertrophied configuration of astrocytes responding to injury. 3.4. Serum cytokine levels As shown in Fig. 6, serum TNF-α concentration significantly increased from a baseline of 135.8 ± 41.3 pg/ml in control rats to a maximum of 263.2 ± 97.7 pg/ml at 4 h after mild injury. There was increase in TNF-α levels at day 1 PI but it was non-significant. Day 3 and day 5 did not show any difference in TNF-α levels as compared to controls. IL-10 levels were significantly increased at day 1 (12.03 ± 3.32 ng/ml) as compared to control (5.65 ± 3.09 ng/ml). No other time point showed any significant difference in IL-10 levels as compared to control. 4. 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., 2013). In our present study, we also report a similar pathway following injury where pro-inflammatory cytokine, TNF-α was found to significantly increase at 4 h PI with enhanced expression of anti-inflammatory cytokine, IL-10 at day 1 PI. Also astrocytes

Fig. 3. Figure showing plots of diffusion measures as a function of mean GFAP+ cells/FOV. Pearson's correlation revealed significant results. (I) MD showed significant inverse correlation with histological count along with (III) AD and (IV) RD, however (II) FA did not show any significant correlation.

Please cite this article as: Singh, K., et al., Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.07.016

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K. Singh et al. / Experimental Neurology xxx (2015) xxx–xxx

Fig. 4. Graph showing quantitative analysis of GFAP+ cells in rat mTBI model. Graph shows mean GFAP+ cells per field view with error bars indicating standard deviation. Cortex showed significantly increased GFAP+ cells at day 3 and day 5 as compared to controls (day 0) shown by asterisk marks.

(GFAP+ cells) with hypertrophied appearance significantly increased at day 3 and day 5 PI further strengthening our hypothesis of mild injury induced inflammatory response. With further speculation of whether these changes translate into diffusivity alterations and could be monitored with non-invasive techniques like DTI, we found that changes in inflammatory markers were accompanied with injury induced reductions in MD and RD values as early as 4 h PI which reached the level of statistical significance at day 3 and day 5 in the cortical region PI. We also observed significant inverse correlation between MD, RD and AD values with mean GFAP count in CTX region. Many studies have tried to establish the pathophysiological mechanism following clinical mTBI and phases of experimental mTBI with attempts to develop a non-invasive method of monitoring these changes. Available literature indicates high concentrations of inflammatory cytokines may be produced by resident microglia and infiltrating monocytes/macrophages during the acute phase of injury (D'Mello et al., 2009). A recent study (Baratz et al., 2015) on mouse mild of closed head injury showed elevated levels of TNF-α till 12 h PI which is in consensus with our study.

The mechanical stress during the traumatic insult to the brain leads to stretching of axons, cells and supporting structures such as oligodendrocytes (Povlishock and Katz, 2005; Browne et al., 2011). Subsequently there is a decline in the function of Na+–K+ ATPase pump, leading to an influx of sodium, calcium, and water (Rosenblum, 2007). The influx of fast moving extracellular water to the diffusion restricted intracellular region results in a net decrease in MD values (Vorísek et al., 2002). Also, trauma induced cytotoxic edema reduces extracellular space, further depleting the regional MD values. Earlier TBI studies (Vorísek et al., 2002) with different injury severity and induction mechanism have shown reduced MD at the injury site (similar with the findings of our study showing reduced diffusion in the cortical gray matter) indicating towards similar pathological mechanism PI in our model. The homeostasis of extracellular and synaptic spaces (energy dependent uptake mechanism) regulating the MD values has also been found to be affected by astrocytic activity in the CNS. Astrocytes are thought to regulate the extracellular concentrations of water, potassium, glutamate and other neurotransmitters (Simard and Nedergaard, 2004). Activation of astrocytes and reactive gliosis are key features observed in many CNS diseases including neurotrauma (Kimelberg and Norenberg, 1994; Norenberg, 1994). GFAP has been used as one of the sensitive marker of glial activation (Bignami, 1991). Increased levels of GFAP have been found in both clinical and experimental severe TBI, reflecting cytokine-induced inflammation leading to activation of astrocytes (Nylen et al., 1996; McIntosh et al., 1998). In our present study, we have found significant increase in GFAP+ cells in the cortical gray matter as compared to controls at day 3 and day 5 PI. We have also found a significant inverse correlation between MD values and mean GFAP count in the cortex region. Microscopic examination indicates star-like hypertrophied configuration of astrocytes that, in part, defines astrocytic activation responding to injury in this region. Based on these findings we speculate that at day 3 and day 5 PI, there may be initiation of reactive astrogliosis leading to hypertrophy of astrocytic processes and increased numbers of reactive glial cells imposing diffusion restrictions in the CNS leading to reduced MD values. These results indicate towards an injury induced astrocytic response which could be predicted via diffusion measures and vice versa. A previous study revealed that astrocyte activation, with consequent morphological alterations such as change in their shape,

Fig. 5. mTBI in rodents increases GFAP immunoreactivity. Representative images (10X-I panel) of rat cortex tissue showing increased GFAP+ cells at day 3 (B) and day 5 (C) post injury as compared to control — day 0 (A). A higher magnification (40× — II panel) image of injured animals from both day 3 and day 5 shows injury induced enhanced astrocytic activation. Scale bar indicates 100 μm.

Please cite this article as: Singh, K., et al., Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.07.016

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Fig. 6. Showing concentrations (pg/ml) of (A) TNF-α and (B) IL-10 (ng/ml) at various timepoints PI and control. mTBI significantly increases TNF-α concentration at 4 h and IL-10 at day 1 after traumatic insult indicating injury induced inflammatory response PI. (D0 is control; D1 is day 1, D3 is day 3, D5 is day 5).

increases in the number of fibrillary processes (Theodosis et al., 2008) and subsequent increase in cell perimeter may lead to a reduction in the RD values which is mainly responsible for reduced MD values (Blumenfeld-Katzir et al., 2011). Various clinical and preclinical studies have shown heterogeneous pattern of decrease in MD, RD and AD values in gray matter region during both acute and chronic phases of TBI (Bouix et al., 2013). A recent study on acute (2–4 h post injury) animal CCI trauma model (Xu et al., 2011) has shown significant decreases in MD and RD values with accompanied decrease in AD values and an increase in FA in the cortical gray matter region with no significant change in the white matter region of CC. Findings of reduced diffusivity in our study are in consensus with the findings of this study. The authors have speculated that gliosis is the major contributor of these changes. They also speculated that factors such as high viscosity from cell debris, elevated lipid content within area of necrosis, and decreased water content within myelin sheaths can limit water diffusivity (Peled, 2007). Other studies on chronic human subjects with persistent post-concussive symptoms in mTBI (n = 11; 62.08 ± 46.35 months post injury) have shown concomitant reductions in AD/RD with MD may be due to more hindered water diffusion in these areas along with neuroplasticities such as increased neurite density (Blumenfeld-Katzir et al., 2011), the presence of plaques or other diffusion hindering material (Baugh et al., 2012). Various animal models of mild neurotrauma have shown decreased diffusivity values (Zhuo et al., 2012; Hylin et al., 2013) along with acute neuroimflammatory consequences. These studies suggest that other in vivo imaging techniques like diffusion kurtosis imaging as well found similar temporal alterations in mTBI as DTI. Animal model of mild FPI showed reduced MD values (Hylin et al., 2013) along with no visible neuronal loss in neocortex or hippocampus region but enhanced GFAP and Iba1 immunorectivity. There was no visible brain contusion (similar to normal conventional MRI in our model) but transient suppression of acute neurological functions. However, in contrast, our model showed no significant alteration in GFAP immunoreactivity in CC and thalamus regions as found in this model. The discrepancies observed in the resulting pathology may be due to experimental methodology, such as surge of blood pressure and craniotomy, severity and mechanism of injury, type of animal model used, and modalities being used to investigate PI changes. Most of the studies employ craniotomy which leads to cerebral trauma excluding cranial trauma which is an important aspect of craniocerebral injuries inflicted in clinical TBI and has been taken into account in our model. Different animal models exhibit different pattern of concussion, contusion, traumatic axonal injury, hemorrhage and skull fracture (Xiong et al., 2013) leading to differential temporal profile of pathology. To conclude, these results indicate our model delivers the noninvasive monitoring of diffusivity alterations indicating pathology pattern using DTI. In absence of any signal abnormality on conventional T2 image of mTBI rodent model, we found successive decreases in MD

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Please cite this article as: Singh, K., et al., Longitudinal changes in the DTI measures, anti-GFAP expression and levels of serum inflammatory cytokines following mild traumatic brain injury, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.07.016