A novel traumatic brain injury model for induction of mild brain injury in rats.

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Contents lists available at ScienceDirect

Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth

Basic Neuroscience

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A novel traumatic brain injury model for induction of mild brain injury in rats

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Tahereh Ghadiri Garjan a,b , Mohammad Sharifzadeh b,c,∗∗ , Fariba khodagholi d , Seyyed Mostafa Moddares Musavi b , Gholamreza Hasanzadeh a , Mohammadreza Zarindast a , Ali Gorji b,e,f,∗ a

School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran Shefa Neuroscience Research Center, Tehran, Iran c Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 9 d Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 10 11 Q2 e Institute of Physiology I, Department of Neurosurgery, Epilepsy Research Center, Münster University, Germany f Institute of Physiology I, Department of Neurology, Epilepsy Research Center, Münster University, Germany 12 7 8

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a b s t r a c t

• Mild TBI constitute approximately

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80% of all traumatic brain injuries in humans. Improved animal models that mimic all aspects mild TBI in humans are needed. A novel stereotaxic coupled weight drop device was designed. The new device induced both primary and secondary damages at trauma site. This new model of TBI is suitable for evaluation of mild TBI.

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Article history: Received 3 April 2014 Received in revised form 26 May 2014 Accepted 28 May 2014

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Keywords: Brain injury Weight drop Dark neurons Head trauma CNS

Background: Due to the marked heterogeneity of human traumatic brain injury (TBI), none of the available animal model can reproduce the entire spectrum of TBI, especially mild focal TBI. This study was designed to develop a modified TBI weight drop model for induction of focal mild cerebral injury. New method: A stereotaxic coupled weight drop device was designed. Principle arm of device carries up to 500 g weights which their force was conveyed to animal skull through a thin nail like metal tip. To determine the optimal configuration of the device to induce mild TBI, six different trials were designed. The optimal configuration of the instrument was used for evaluation of behavioral, histopathological and molecular changes of mild TBI. Results: Neurologic and motor coordination deficits observed sharply within 24 h post injury period. Histological studies revealed a remarkable increase in the number of dark neurons in trauma site. TBI increased the expression of apoptotic proteins, Bax, BCl2 and cleaved caspase-3 in the hippocampus. Comparison with existing methods: Our designed TBI device is capable to produce variable severity of TBI from mild to severe. The main advantage of the new TBI model is induction of mild local unilateral brain

∗ Corresponding author at: Epilepsy Research Center, Universität Münster, Robert-Koch-Straße 27a, D-48149 Münster, Germany. Tel.: +49 251 8355564; fax: +49 251 8355551. ∗ ∗ Corresponding author at: Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. E-mail address: [email protected] (A. Gorji). http://dx.doi.org/10.1016/j.jneumeth.2014.05.035 0165-0270/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Garjan TG, et al. A novel traumatic brain injury model for induction of mild brain injury in rats. J Neurosci Methods (2014), http://dx.doi.org/10.1016/j.jneumeth.2014.05.035

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injury instead of traumatization of the whole brain. This model does not require craniotomy for induction of brain injury. Conclusion: This novel animal TBI model mimics human mild focal brain injury. This model is suitable for evaluation of pathophysiology as well as screening of new therapies for mild TBI. © 2014 Published by Elsevier B.V.

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1. Introduction Numerous events, such as falls, sports, motor accidents and military injuries, can lead to traumatic brain injury (TBI). TBI is a leading cause of mortality and morbidity in the population below 50 years with a peak incidence in young adults (Bruns and Hauser, 2003; Marklund and Hillered, 2011). More than 1.5 million people worldwide die due to TBI each year. The number of TBI victims globally raised sharply in the last few decades (Tagliaferri et al., 2006). TBI can cause long-term physical disability, neurobehavioral deficits and disrupt quality of life (Masel and DeWitt, 2010). Mild TBI constitute approximately 80% of all traumatic brain injuries in humans (Jennett, 1998). The deficits produced by mild TBI are frequently more subtle, less often recognized, and more contentiously debated than are those resulting from severe TBI (Dikmen et al., 2001). In spite of the developments in the prevention, diagnosis and management of TBI, there is still no adequate treatment available to ameliorate its disabling effects. In view of the heterogeneous nature of the clinical situation in TBI, numerous animal models of such injury have been created to study the underlying mechanisms of human TBI. The purpose of experimental models of TBI is to replicate certain pathological components or phases of clinical trauma in experimental animals aiming to address pathophysiological mechanisms and develop preclinical therapeutics (Marklund et al., 2006; Marmarou and Povlishock, 2006). The major techniques of experimental TBI models are including the controlled cortical impact, weight-drop, impact acceleration and fluid percussion models (Morales et al., 2005; Cernak, 2005). A variety of morphological, cellular, molecular, bioelectrical and functional changes of human TBI, including alterations in ionic homeostasis, generation of free radicals, eliciting neuroinflammatory responses, releases of excitatory amino acids, initiation of negative DC shifts (spreading depolarization) and changes in neurotransmitter systems have been characterized by various experimental TBI models (Finnie and Blumbergs, 2002). However, there is no single animal model of TBI that can reliably mimic all aspects of human TBI, especially mild focal TBI (Morales et al., 2005; Cernak, 2005; Finnie and Blumbergs, 2002). Improved animal models that reflect the relevant processes in mild TBI in humans, and in which new novel biomarkers might be identified and evaluated, are needed (Zetterberg et al., 2013). Traditional weight drop TBI models, such as impact acceleration model, have some disadvantages and limitations. Due to lack of enough control on forces induced by a weight drop, these models frequently destroy the majority part of the ipsilateral cortices and lead to central respiratory depression. Different weight-drop models usually require craniotomy or have a high probability of skull fracture and rebound injury. The results obtained by these models clearly not comparable with the extent of brain injuries observed in many of survivors of human TBI, especially those suffered from mild-to-moderate TBI. It is suggested that if TBI could be induced without craniotomy or skull fracture, it will be more ideal for studying human TBI (Morales et al., 2005). Cortical compact injury model allows for better control over mechanical factors, such as velocity of impact and depth of resulting deformation but is not able to accurately mimic bioelecterical and functional aspects of human TBI (Morales et al., 2005; Cernak, 2005). In order to fulfill the needs for a reliable model

of focal TBI and cost effective device, the present study introduces a new method of focal brain injury via a novel weight drop TBI model.

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2. Materials and methods

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2.1. Apparatus arrangement

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The overall arrangement of the new TBI device and its accessory components are shown in Fig. 1. The apparatus consists of: 1. Weights with various sizes and desired mass (40, 80, 100 and 200 g). The delivered force can be changed by assembling the different weights on to dropping arm (Fig. 1A–D). 2. Dropping arm which is relied on a metal rod in free end of a magnetic stand at the basement of apparatus. This part carries desired number of weights in longitudinal fashion. The maximum weight it can tolerate is 600 g (Fig. 1B). 3. Basement of device which is made from heavy metal disk (5 × 5 cm) with magnetic property. Another metal rod (25 cm length) is projected from the central part of the basement. This part and basement are fixed part of the device. The angle between the metal rod and the dropping arm can be adjusted (30–120◦ ) and provides diverse dropping heights. In this meet point, electrical energy converts to driving force of dropping arm falling, by pressing the button of trigger handle (Fig. 1A–C). 4. Multiple size tips (4 tips, various diameters in the encountering side, max area 10 mm2 ), which can screw to a hammer like metal rod (7 cm length) connected to free end of dropping arm with a 90◦ angle (Fig. 1D–H). 5. Two connectors; one for supplying electricity source and have a trigger button, another one for connecting to oscilloscope (Fig. 1A). 6. A stereotaxic surgery device (Fig. 1A). It should be mentioned that the height which weight launches from, varies depending on the angle between the principle arm and an assumptive horizontal line. In our method weight is not launched directly into skull and the force conveys through vertically dropping of hammer like metal rod due to release of principle arm by trigger button. Launching of the dropping arm is controlled via pressing button of trigger connector. The force of weights transmits to the skull through tip of the hammer like metal rod. The force of dropping weight on the head of animal was measured by a force sensor (FSG15N1A, Honeywell, Germany; sensitivity 0.24 mV/g, measurement range 0–1500 g) located on the tip of hammer like metal rod. The signal from the sensor is amplified by a homemade amplifier (1 V/kg) and the pressure pulse of the injury was recorded on a storage oscilloscope triggered by the fall of the dropping arm. The amplitude of the pulse was used to determine the intensity of the weight drop force. Two experimental protocols were conducted. The first one was aimed to determine appropriate weight, angle and height to induce a mild-to-moderate focal TBI. Using histopathological and molecular investigations, the second series of experiments were designed to confirm induction of brain injury induced by the new TBI device.


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Fig. 1. (A) Overall arrangements of the new designed traumatic brain injury (TBI) apparatus. (1) stereotax, (2) weight drop device and (3) oscilloscope. (B) Device perior to induction of TBI, two main parts of the basement of apparatus are shown: (1) metal rod and (2) heavy metal disk with magnetic property. (C) Apparatus posture after induction of TBI. (D) Different parts of principle part of device including (1) tip, (2) hammer-like metal rod, (3) dropping arm and (4) weights are shown. (E) Oscilloscope. (F) Fixed head after trauma. (G) Photograph of a typical brain after brain trauma. Trauma was conducted on parietal cortex between −3.2 and −4.00 from bregma. (H) Different size tips.

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2.2. The first experimental protocol In the first protocol, 36 male Wistar rats (230–300 g) underwent TBI through different trials. In each trial, the angle, weight and height were manipulated to optimize the induction of mildto-moderate TBI. During these trials different weights (160, 350 or 500 g) dropped from various heights (30 or 40 cm) with different angles (60◦ or 75◦ ) as well as tips (2 or 4 mm2 ) and with or without skull exposure (Fig. 1; Table 1). The animals were deeply sedated with a 1:1 mixture of ketamine (Sigma; 100 mg/ml) and xylazine (Rompun® , Bayer; 20 mg/ml (Yuan et al., 1988), at a dose of 1 ml/kg of body weight (intraperitoneally) throughout the surgical procedures and TBI. Surgical areas were shaved and antiseptically cleaned prior to positioning in a stereotaxic frame. In all trials except the first one, skull was exposed by a midline longitudinal scalp incision and the fascia cleared to expose the surface of the skull. The head was fixed and major arm of device content weight was released and force was conveyed on the skull from a desirable height. Depending on the size of selected tip and dropped height, blunt or penetrated TBI was induced. Neurologic examination were performed before TBI as well as 24 h, 48 h, 7 days and 14 days after head injury using modified Neurologic Severity Score (mNSS) test (Fig. 1). Modified mNSS test, which is sensitive to unilateral cortical injury, consists of tasks on motor function, alertness, vestibule-motor functions as well as other physiological behaviors from 0 to 18 grade, whereby 1 point is awarded to failure of one task and no points are given for success. Thus, the higher score indicates the more severe injury (for instance 18 points means severe neurological dysfunction, with failure at

all tasks and 0 score shows normal function (Xiong et al., 2010). All rats were trained and pretested prior to injury. Results of mNSS test following the first trial showed that the amount of trauma did not lead to neurologic deficit. Therefore, during the next three trials, we gradually increased the weights from 160 to 500 g along with exposure of skull by a midline longitudinal scalp incision prior to weight drop. Except the first trial all weights were dropped on the middle of the right parietal bone. 2.3. The second experimental protocol This part of study was conducted on 69 male Wistar rats weighting between 230 and 300 g. All animals were kept under 25 ◦ C ambient temperature and 12 h cycle of light per day. After scalp exposure, rats were subjected to TBI and then randomly assigned to undergo decapitation 1 h (n = 20), 3 days (n = 20) or 14 days after TBI (n = 20). Sham control animals (n = 9) went through the same surgical procedures without TBI. Animals were deeply anesthetized; the head was fixed in stereotaxic apparatus (Stoelting Instruments, USA) and major arm of device content 500 g weight was released from a height of 30 cm on exposed right parietal bone. After trauma, the rats received supporting oxygenation during recovery. Survived animals were returned to their home cages and allowed simplified access to food and water 2 h after induction of TBI. 2.3.1. Open field behavioral assay Post-injury locomotor and exploratory activities were assessed by the open field behavioral test. The open field area consisted of an empty 50 × 50 cm2 quadrant, divided by painted blue lines into 25


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squares and surrounded by 25 cm height walls to prevent animal from escaping (Curzon et al., 2009). Tests were performed at various time intervals before TBI and 24 h, 48 h, 7 as well as 14 days after weight drop injury and sham surgery. Each rat was placed in the center of open field and videotaped for 5 min. The grooming time and the number of rear and locomotion (assessed as the number of squares entered by the nose and two forepaws) over the 5-min testing period were measured. 2.3.2. Histological assessment Animals were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) (Marmarou and Povlishock, 2006) and decapitated after 1 h, 72 h and 14 days after injury. Brains were rapidly taken and post-fixed in 4% formalin and processed for paraffin embedding. The appearance of neurons with condensed darkly stained nuclei and bright cytoplasm (dark neurons) was used to assess neuronal injury following TBI (Sadeghian et al., 2012). To visualize dark neurons, histological features of neuronal injury were stained with cresyl violet (Nissl staining). Three noncontiguous 8-␮m coronal sections were taken between −3.2 and −4.00 from bregma (Lowenstein et al., 1992) and stained using a 0.3% solution of cresyl violet and toluidine blue. Digital images of the ipsilateral and contralateral parietal cortex, hippocampal CA1 and CA3 regions as well as the entorhinal cortex were captured at 40× magnifications (Olympus, Japan). An equivalent slice through the cortex and hippocampus from both the ipsilateral and contralateral hemispheres were analyzed. A single-blinded investigator counted dark neurons with shrinkage in cell bodies and diffusion of nucleus by physical dissector counting rule in each delineated region (Sadeghian et al., 2012). 2.3.3. Lesion size measurement The brains immediately or 24 h after TBI were cryosectioned from caudal to rostral with 1 mm thickness and stained by cresyl violet and triphenyltetrazolium 2% (TTC; Sigma, St. Louis, USA) to evaluate lesion volume (Kramer et al., 2010). The Nissl and TTC stained specimens spaced 100 ␮m apart between −3.2 and −4 bregma were photographed at 1.25 magnifications by canon eyepiece magnifier digital among traumatized brains which had obvious contusion and analyzed by Image J 1.41o for lesion volume analysis. Total lesion volume was calculated using Cavalieri’s formula [Volume = A × t × ISF] where A = the lesion areas; t = section thickness and ISF = inverse of the sampling fraction (1 in 10 sections was counted, i.e. sampling fraction = 1/10 (Sashindranath et al., 2012). 2.3.4. Western blot analysis Three rats of each group (investigated 1 h, 3 days or 14 days after TBI) were selected for western blot analysis. The brains were cut coronally by vibratome and hippocampal tissues were manually dissected and proteins extracted by homogenization in 4 ◦ C in 50 mM Tris–HCl buffer, pH 7.4, containing 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml pepstatin and 1 mg/ml leupeptin. Samples were centrifuged at 10,000 rpm (4 ◦ C) for 5 min and protein concentrations of centrifuged supernatants were determined by the Bradford test. Samples were diluted at 0.5 ␮g/␮l in 0.25 M Tris–HCl buffer, pH 6.8, 10% glycerol, 10% sodium dodecyl sulfate (SDS), 10 mM dithiothreitol (DTT), 0.0025% bromophenol blue. Protein samples were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore). The PVDF membranes were blocked for 75 min in TBS-T buffer (100 mM Tris–HCl; 0.9% NaCl, 0.1% Tween 20, pH 7.4) containing 5% non-fat dry milk. Blots were then incubated with rabbit monoclonal anti-Bax rabbit monoclonal, bcl2 rabbit polyclonal and caspase-3 rabbit monoclonal antibodies (1:1000, Santa Cruz,

Fig. 2. Neurologic functions of rats according to modified Neurologic Score test (mNSS) following traumatic brain injury (TBI) in various trials (1–6). Severity of trauma during the first and second trials did not affect neurologic performance. Enhancing the weights and manipulating other variables, score of mNSS was significantly raised after TBI and reached to the maximal point 24 h after TBI in trials 3, 4 and 6. In trial 5, all rats were dead after TBI and it was not possible to test the neurlogic function. During the next days neurologic function slowly improved and reached to the value of pre-TBI by day 14. Values represent mean ± SEM. ***, ** and * indicate P < 0.001, P < 0.01, P < 0.05, respectively. Values were compared to sham rats.

Gemany) overnight at 4 ◦ C. Anti-␤ actin rabbit monoclonal antibody (1:1000, Sigma, St. Louis, USA) was used to detecting actin as internal control after stripping. Filters were washed with TBST buffer 3, each time for 15 min after primary antibody and then incubated for 1–2 h with secondary antibodies (horseradish peroxidase conjugated gout anti-rabbit; 1:3000; Santa Cruz, Germany). Immunoreactivity was revealed chemically by ECL (Amersham Biosciences, Freiburg, Germany). The blots were exposed to X-ray film sensitive to blue light for 5–30 s. The developed films were scanned on Bio-Rad scanner. Densitometric analysis was carried out by the monomeric bands data for quantitation with Image J software. The experiments were carried out in accordance with the EU Directive 2010/63/EU on the protection of animals used for scientific purposes and were approved by the local ethical committee of Shefa Neuroscience Center.

2.4. Statistical evaluation Statistical analysis was performed using software SPSS 16. All the values were expressed as mean ± SEM. Multiple comparisons were performed using ANOVA followed by Tukey test. A p-value of 0.05 or less was considered significant.

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To identify the proper adjustment of the instrument, six different trials under diverse circumstances were performed. The conditions of different trials of TBI induction and outcomes of each trial are summarized in Table 1. TBI produced by trails 5 was lethal and all rats that traumatized by these trials died within 0–60 min after the trauma. Trials 1 and 2 did not induce any fracture, bleeding or neurological deficits after TBI. Trial 5 produced penetrant head injury with pore like fracture and brain contusion. Trials 4 and 6 produced the optimal results for induction of focal mild-tomoderate TBI with significant neurological deficits 6 h after TBI and the lowest fatality (Table 1).


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3.2. Modified Neurological Severity Scores Trials 1 and 2 did not induce any neurological deficits within 14 days of TBI. The highest mNSS score was observed 24 h after TBI in rats traumatized by trials 3, 4 and 6 (P < 0.001). However, the neurological deficits improved after 48 h of TBI in these rats and returned to pre-TBI conditions 2 weeks after TBI. Trial 3 induced the most significant neurological deficits compared to other trials after 24 h, 48 h and 7 days of TBI (Fig. 2). Sham operated rats did not show any neurological deficit.

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3.3. The second experimental protocol According to the first protocol in our study, trial 4 (500 g weight, 30 cm distance, 65◦ angle) was accompanied by the lowest rate of mortality and penetrating head injury. After exposure of skull to a single impact with trial 4 (n = 60), rats were mostly in a fully conscious state and showed normal grooming and social interactions with their cage mates during 24–48 h after TBI (n = 59). Although two rats were suffering from apnea after TBI in this trial, oxygenation and cardiac massage was effective in restoring the normal respiration within 2–3 min. Therefore, trial 4 was selected for functional and histopathological investigations. 3.4. Motor activity in open field test In the open field test, three parameters (including the number of locomotion and rearing as well as grooming time) were evaluated 24 h, 48 h, 7 days and 14 days after TBI or scalp exposure (sham group). Sham operated animals did not show any changes in ambulation events or rearing and grooming after surgery (Fig. 3A–C). The mean number of locomotion activities decreased 24 h after TBI and returned to the values of pre-TBI state after 48 h of head trauma (P < 0.05; Fig. 3A). In contrast, TBI rats showed a significant reduction in the number of rearing activities 24 h as well as 48 h after trauma compared to sham rats (P < 0.001; Fig. 3B). The decreased rearing activities improved after 48 h and returned to its normal level 14 days after TBI. Grooming time in TBI rats increased during the first 24 h after TBI (P < 0.001; Fig. 3C) and returned to pre-TBI values after another 24 h of TBI. 3.5. Number of dark cell Darks neurons were recognized by neuronal shrinkage, pyknosis and surrounding spongiosis (Jafarian et al., 2010). The mean number of dark cells was evaluated 1 h, 72 h and 14 days after TBI in the hippocampal CA1 and CA3 areas as well as in the entorhinal and parietal cortices, in both ipsi- and contralateral hemispheres to the trauma site (Fig. 4A and B). The mean number of dark neurons in different brain regions of the contralateral hemisphere to TBI site was not affected by brain trauma compared to sham rats (data not shown). TBI significantly increased the mean number of dark cells in the parietal and entorhinal cortices as well as hippocampal CA1 and CA3 areas of the ipsilateral hemisphere to TBI site after 72 h and 14 days of TBI compared to sham group (P < 0.001). The number of dark neurons 14 days of TBI in the parietal cortex, however, significantly reduced compared to 72 h after TBI (P < 0.01; Fig. 4A and B).

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Table 1 Different set up of TBI device and observed outcomes of 6 trials of first experimental protocol. Modified Nerulorologic Severity Score, mNSS; RH, right hemisphere; RPB, right parietal, * tip with barrier.

Impact force(N)

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TBI caused a brain lesion in the ipsilateral somatosensory cortex to TBI site in 25% of traumatized rats. Mean lesion volume was 14.3 ± 3.4 mm3 after 1 h (n = 6). This volume significantly increased to 18.7 ± 6.12 mm3 after 72 h of TBI (P < 0.001; n = 5) but decreased to 13.7 ± 3.1 mm3 when investigated 14 days after TBI (P < 0.01;


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Fig. 3. Performance of open field test to evaluate the behavioral effect of traumatic brain injury (TBI) induced by the new designed device. Behavioral test was performed before, 24 h, 48 h, 7 days and 14 days after TBI using open field test. (A) The mean number of line crossing movements was reduced 24 h after TBI and returned to pre-TBI level 48 h after TBI. (B) The mean number of rearing movements significantly decreased in the first 48 h after TBI and returned to pre-TBI level 7 days after TBI. (C) The mean duration of grooming behavior significantly increased 24 h after and returned to pre-TBI level after another 24 h after TBI. Values represent mean ± SEM. * and *** indictae P < 0.05 and P < 0.001, compared to sham rats.

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n = 5; Fig. 5G). Typical cresyl violet- (Fig. 5A–D) and TTC (Fig. 5H–I) stained photomicrographs of coronal sections of the somatosensory cortex of sham and TBI (1 h, 72 h and 14 days) rats are shown. There was no lesion in contralateral cortex to TBI site as well as in sham rats. 3.7. Expression of anti and pro apoptotic factors following TBI Using western blot techniques, the expression of Bax, Bcl2, cleaved capsase-3 and pro-caspase-3 in the ipsilateral hippocampus to the brain trauma site was evaluated 1 h, 72 h and 14 days after TBI. The expression of these factors was not affected by trauma 1 h after TBI. However, the Bax/bcl2 ratio and both expression of cleaved and procaspase-3 were significantly increased 72 h after TBI compared to sham group (P < 0.001; Fig. 6A–D). The expression of these factors significantly decreased 14 days after TBI; still remained significantly higher than sham rats (P < 0.01). The caspase-3/␤-actin values were also significantly increased 72 h after TBI and reduced 14 days after TBI (Fig. 6C). 4. Discussion Primary brain injury is reflected to early manifestation of direct mechanical force which may be local (such as contusion) or diffuse (like diffuse brain injury; (Kemp et al., 2003; Dixon et al.,

1991; Yamaki et al., 1997). Secondary brain injury arises from consequently activation of various pathophysiological processes after acute phase of TBI (Xiong et al., 2013; Werner and Engelhard, 2007). The present study indicated that trauma induced by our modified weight drop method produced a local TBI, which resulted in not only primary injury immediately after the trauma but also secondary damages within next few days. Occurrence of secondary injury with our modified weight drop TBI model confirmed using histological, molecular and neurologic evaluations. Both primary and secondary damages were observed only at trauma site and nearby vicinity.

4.1. New weight drop method and induction of brain injury The purpose of experimental models of TBI is to replicate pathological components of human brain trauma in animals aiming to address pathology and/or treatment. A variety of functional, molecular, cellular and morphological alterations observed in human TBI have been identified in the brain tissue of animals traumatized by our modified weight drop model of TBI (Cernak, 2005; O’Connor et al., 2003; Walker and Pickett, 2007). Given the large number of individuals that experience mild-tomoderate TBI annually, it is indeed fortunate that the majority of these patients recover fully within the first year after TBI. The development of neurological deficits is predicated on a complex


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Fig. 4. The effects of traumatic brain injury (TBI) on the mean number of dark neurons in the parietal cortex (PC), ectorhinal cortex (ECT) as well as CA1 and CA3 subregions of the hippocampus of ipsilateral hemisphere to TBI site, in sham rats as well as 1 h, 72 h and 14 days after TBI by the new designed device. (A) Light-microscopic appearance (40× magnification) of coronal sections of the rat brain in sham and TBI (1 h, 72 h and 14 days after trauma) rats stained with cresyl violet. (B) The mean number of dark neurons in different brain regions of sham and TBI (1 h, 72 h and 14 days after trauma) rats indicates an increase of dark neurons in PC, ECT and CA3 hippocampal area 72 h after TBI. Note that highest rate of dark neurons is near to trauma site in PC. The mean number of dark neurons was compared between experimental groups and analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests. Values represent mean ± SEM. # and * indicate P < 0.01 and P < 0.001, respectively.

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set of factors, such as neural injury induced by mild TBI, preexisting and/or comorbid post-traumatic psychiatric diseases and occasionally on conscious and/or unconscious efforts to obtain primary and secondary gains (MacMillan et al., 2002; Wood, 2004; Arciniegas et al., 2005). Mild-to-moderate type of TBI in our study (trial 4) led to a transient impairment of neurologic functions and locomotor activities; reduction of rearing activities and prolongation of grooming behavior. The neurologic and motor impairments observed after TBI gradually improved in the first week and completely returned to pre-TBI value on day 14. In keeping with our data, experimental data showed significant sensorimotor and neurologic deficits in hanging wire test from day 1 to day 7 after TBI which were recovered close to preinjury baselines by day 21 (Zhao et al., 2012; Aligholi et al., 2014).

Using computed tomographic scanning, it has been shown that mild TBI induced brain abnormalities in 20–35% of patients with GCS scores of 13 or 14 (Stein and Ross, 1992; Harad and Kerstein, 1992). Investigations using data acquisition and interpretation methods revealed significant post-traumatic cerebral structural abnormalities, including neocortical and subcortical atrophy, ventricular dilation and white matter injury in mild TBI (Bigler, 2003; Arciniegas and Silver, 2001). Our data indicated TBI induced by trial 4 resulted in formation of visible lesion in 25% of traumatized rats. In line with our results, some other experimental TBI model reported similar cortical cell loss directly beneath the impact site and formation of brain lesion (Ates et al., 2007; Flierl et al., 2009). A number of complex molecular pathologies contribute to mild TBI in humans; these are including metabolic changes and oxidative


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Fig. 5. The mean volume of the cortical lesion induced by traumatic brain injury (TBI) using the new designed device. (A–D) Representative (1.25× magnification) photomicrographs of cresyl violet stained coronal sections of sham rats (A) and TBI rats 1 h (B), 72 h (C) and 14 days (D) after head trauma. (E–G) Representative (10× magnification) photomicrographs of cresyl violet stained coronal sections of TBI rats 1 h (E), 72 h (F) and 14 days (G) after TBI. (J) A graph illustrated the volume of traumatic brain lesion in sham and TBI (1 h, 72 h and 14 days after trauma) rats. Values compared betwee sham and TBI rats. Data were expressed as mean ± SEM. ** and *** indicate P < 0.01 and P < 0.001, respectively.

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stress, neuroinflammation, vascular dysfunction, altered cellular adhesion as well as neuronal and glial injury and/or loss. These alterations are central, if perhaps transient, neuropathological features of mild TBI in humans (Povlishock and Jenkins, 1995). Experimental models of TBI are of vital importance in the identification of the complex mechanisms leading to both necrotic and apoptotic neuronal death after brain injury. Our study demonstrated that TBI induced by the new designed device triggered reversible cell damage. According to our study, the number of dark neurons significantly reduced on day 14 in all regions compared to acute phase of TBI. Dark neurons represent various states of neuronal damage in brain insults, which could terminate to death or survival (Ishida et al., 2004). Clinical studies have reported

enhancement of apoptosis modulator levels, such as Bcl2, in cerebrospinal fluid of patients suffering from TBI (Uzan et al., 2006). In our study, enhancement of apoptosis modulator levels was observed first 3 days after TBI. The expression of these factors reduced 14 days after TBI. Detectable changes in the level of protein expression, due to nature of protein synthesis system need more than 1 h. There are similar results in human traumatic brain injury (Petzold et al., 2011; Raghupathi, 2004). It is not surprising that the amount of Bax, Bcl2 and cleaved caspase-3 did not show significant increase 1 h after TBI. The level of Bcl2 protein, cleaved caspase 3 and DNA fragmentation showed increase in the brain of TBI patients at least 1 day after injury (Clark et al., 1999). The abovementioned evidence suggests that the new designed weight


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Fig. 6. Alteration in expression of apoptotic proteins in the hippocampus of rats with traumatic brain injury (TBI) and sham rats. (A–D) TBI significantly raised the ratio of Bax/Bcl2 and cleaved casepse-3 within 72 h and 14 days after TBI. (B, C) The Bax/bcl2 ratio as well as caspase-3/␤-actin values were also significantly increased 72 h after TBI and reduced 14 days after TBI * represents significancy compared to sham. Data were represented as mean± SEM. # and *** indicate P < 0.01 and P < 0.001, respectively.

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drop model is an improved animal model of TBI, which reflects the relevant processes in mild TBI in humans.

4.2. Advantages of modified weight drop model The present weight drop model has plentiful advantages in comparison to previous TBI models. (i) Craniotomy before induction of TBI makes animal model different from human TBI. The presented TBI model does not require craniotomy before trauma and can produce histological and molecular changes similar to human TBI. This makes this model safer with less animal loss compared to other focal weight drop models (Ates et al., 2007). (ii) Our designed TBI device is capable to produce variable severity of TBI from mild to severe (the first experimental protocol). By manipulation of the location of weight on principle arm and amount of weights in addition to the launched angle, it is possible to adjust the delivered energy and induce different levels of TBI. Although this apparatus designed for mild focal TBI, severe TBI with extensive bone fracture, contusion and hemorrhage could be induced by weights over 500 mg and height more than 30 cm with thin tip (Table 1). (iii) Another advantage of our model compared to other experimental TBI devices (Ates et al., 2007; Marmarou et al., 1994) is that the imported energy easily could be quantified by connecting the device to an oscilloscope. Giving fixed amount of force (following quantified measuring) could control the range of forces for induction of any level of TBI (mild to severe degrees) and enhanced the reliability and reproducibility of experiments. (iv) This device provides a cost effective solution for induction of TBI in rodents.

(v) The new TBI instrument has the capability to use as a cortical compact injury device by reduction of the size of effector tip or performing craniotomy before injury (trial 5, Table 1). 5. Conclusion Taken all together, histological, behavioral, neurological and molecular data revealed that the presented modified weight drop model mimics mild brain injuries in humans. The advantage of this modified model compared to the previous ones includes induction of TBI without craniotomy, flexibility in changes of the severity of trauma, measurement of the exact force of TBI and production of local brain trauma without involvement of whole brain. This new model of animal TBI is suitable for evaluation of pathophysiology as well as screening of new therapies for mild TBI. Conflict of interest The Authors declare that there is no conflict of interest. Acknowledgements Authors would like to thanks Thomas Westhoff, Lüdger Sasse and Manfred Daweke for their technical assistance. This work was funded by the Shefa Neuroscience Center grant related to Dr. Thesis Q4 25604. References Aligholi H, Hassanzadeh G, Azari H, Rezayat SM, Mehr SE, Akbari M, Attari F, Khaksarian M, Gorji A. A new and safe method for stereotactically harvesting neural stem/progenitor cells from the adult rat subventricular zone. J Neurosci Methods 2014;225(March):81–9.


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A novel model of mild traumatic brain injury for juvenile rats.

Mild traumatic brain injury.

Mild traumatic brain injury.

Sex matters: repetitive mild traumatic brain injury in adolescent rats.

Advanced neuroimaging of mild traumatic brain injury.

Mild traumatic brain injury: neurosensory effects.

Autonomic Dysfunction after Mild Traumatic Brain Injury.

A simple rat model of mild traumatic brain injury: a device to reproduce anatomical and neurological changes of mild traumatic brain injury.

Cervical Spine Involvement in Mild Traumatic Brain Injury: A Review.

Photosensitivity in mild traumatic brain injury (mTBI): a retrospective analysis.

The potential for DHA to mitigate mild traumatic brain injury.

Proposed objective visual system biomarkers for mild traumatic brain injury.

Neurobehavioral Outcomes of Mild Traumatic Brain Injury: A Mini Review.

The spectrum of neurobehavioral sequelae after repetitive mild traumatic brain injury: a novel mouse model of chronic traumatic encephalopathy.

Identification of serum microRNA signatures for diagnosis of mild traumatic brain injury in a closed head injury model.

Resuscitation speed affects brain injury in a large animal model of traumatic brain injury and shock.

A Novel Computer Oculomotor Rehabilitation (COR) Program for Mild Traumatic Brain Injury (mTBI).

A pre-injury high ethanol intake in rats promotes brain edema following traumatic brain injury.

Current status of fluid biomarkers in mild traumatic brain injury.

The Role of Thalamic Damage in Mild Traumatic Brain Injury.

Clinical correlates of retrograde amnesia in mild traumatic brain injury.

Extracellular ezrin: a novel biomarker for traumatic brain injury.

Time-dependent effects of CX3CR1 in a mouse model of mild traumatic brain injury.

Advances in imaging explosive blast mild traumatic brain injury.