An experimental protocol for mimicking pathomechanisms of traumatic brain injury in mice
© Albert-Weißenberger et al; licensee BioMed Central Ltd. 2012
Received: 30 January 2012
Accepted: 2 February 2012
Published: 2 February 2012
Traumatic brain injury (TBI) is a result of an outside force causing immediate mechanical disruption of brain tissue and delayed pathogenic events. In order to examine injury processes associated with TBI, a number of rodent models to induce brain trauma have been described. However, none of these models covers the entire spectrum of events that might occur in TBI. Here we provide a thorough methodological description of a straightforward closed head weight drop mouse model to assess brain injuries close to the clinical conditions of human TBI.
Keywordsclosed head injury traumatic brain injury neurobehavioural deficits astrocyte microglia neurons
Traumatic brain injury (TBI) is a result of an outside force causing immediate mechanical disruption of brain tissue and delayed pathogenic events that can exacerbate the injury (reviewed by ). It represents a leading cause of death and disability in the industrialized countries [2, 3] and a growing health problem in the developing countries [4–7]. To better understand the pathological mechanisms underlying TBI and to develop strategies and interventions to limit the secondary damage, the use of rodent models is essential. A number of rodent models to induce brain trauma have been described; however, none of them covers the entire spectrum of events that might occur in TBI . As an example, the cortical cryolesion model is particularly suited for investigating TBI-associated focal lesions with blood-brain barrier leakage and vasogenic brain edema [9–13] but contre coup and diffuse axonal injuries that typically complicate human head injuries  are missing. This pathophysiology is present in the weight drop models of TBI which use the gravitational forces of a free falling weight to produce a mix of focal and diffuse brain injury [14–16]. One main characteristic of TBI associated diffuse axonal injury is the axonal disruption caused by shearing forces. Typical pathological changes include axonal swelling, axoplasmic ovoid retraction balls and expression of amyloid beta peptides . The original weight drop model in rats by Feeney [18, 19] was optimimized to produce an open-head brain injury whereas the model developed by Shohami [20–22] produces a closed-head brain injury with both focal and diffuse injury and enables its adaptation for mice. We provide here a methodological description of our modification of the Shohami model [14, 21] to induce closed head weight drop injury in mice.
All experiments require an appropriate animal experimentation facility and need to be conducted in accordance with the laws and regulations of the regulatory authorities for animal care. The animal experiments presented here were approved by and conducted in accordance with the laws and regulations of the regulatory authorities for animal care and use in Lower Franconia. A total of 20 male C57BL/mice (10-16 weeks) with a body weight of > 20 g mice were used.
2.1 Weight drop model - Surgery
As the impact can result in trauma-induced respiratory depression and death, posttraumatic oxygen was immediately applied. Then the scalp wound was closed by standard suture material (3-0 Ethilon) and the wound area was treated with lidocain cream. Mice were returned to cages immediately at the end of the surgical procedures where access to water and food is freely available.
Key readout parameters include the assessment of neurobehavioral outcome, activation of resident cells, neurodegeneration, and morphological changes.
2.2.1 Neurobehavioral outcome
During the last years, an extensive number of studies have been accomplished showing that human TBI are associated with physical and cognitive deficits (for a recent review see ). In parallel to human TBI, the brain injury caused by the weight drop model presented here is also associated with neurobehavioral deficits . The neurobehavioral status of mice was obtained by the neurological severity score (NSS) (adapted from ). It consists of 10 individual clinical parameters, including tasks on motor function, alertness and physiological behaviour. Each parameter is described below.
A) Exit circle
B) Seeking behavior
The mouse was placed on the platform and the exploring of the environment and sniffing behavior was monitored. While healthy mice explored the environment (0 points), injured mice do not display this physiological behaviour (1 point).
Monoparesis or hemiparesis was represented by failure to use one (monoparesis) or two (hemiparesis) paws. When being picked up by the tail, a healthy mouse intrinsically gripped a small forceps touching its paws and holds on to it (0 points). If the mouse failed to grip there was given 1 point.
D) Straight walk
The mouse was placed on an even surface and alertness, initiative and motor ability to walk straight was assessed. If a mouse failed to walk straight due to missing initiative or dragging of one or several paws 1 point was given.
E) Startle reflex
A healthy mouse reacted with a bounce and/or wince to a sound stimulus (hand clapping) (score of 0). If the mouse failed to respond 1 point was given.
F) Beam balancing
The mouse was placed on a beam of 7 mm × 7 mm (Figure 3b). Healthy mice are able to balance on the beam for at least 10 s. If a mouse failed to balance, 1 point was given.
G) Beam walk
Motor coordination and balance was also evaluated by the ability of a mouse to transverse a graded series of beams (Figure 3a). If a mouse failed to cross the 3-cm-wide and 30-cm-long beam, 3 points were scored and the test stopped. If a mouse managed to cross the 3-cm beam, then the test was repeated using a beam with a width of 2 cm. For a mouse failing to cross the 2-cm- beam, 2 points were scored and the test stopped. If a mouse managed to cross the 3-cm and 2-cm beam, but failed to cross the 1-cm beam, 1 point was given. Healthy mice managed to cross all beams (0 points).
H) Round stick balancing
2.2.2 Immunhistochemical characterization
Studies characterising the posttraumatic response of resident brain cells in humans, show that TBI leads to activation of astrocytes, microglia, and neurodegeneration [24–27]. Similarly, brain injury induced by the weight drop model presented here initiates pathological processes leading to cellular changes in glia and neurons.
To detect activation of glia immunohistochemical stainings were performed as previously described . Briefly, 10-μm thin brain cryosections were prepared using a cryostat (Leica, Bensheim, Germany). The immunohistochemical staining with antibodies for the detection of glial fibrillary acidic protein (GFAP)-expressing astrocytes (rabbit, 1:500, anti-GFAP; Ab7260; Abcam, Cambridge, UK) and activated microglia/macrophages (rat, 1:100, anti-CD11b; MCA7; Serotec, Raleigh, USA) was performed following standard methods using an avidin-biotin system (Vector Laboratories, Burlingame, USA) and 0.02% diaminobenzidine as chromogen (Kem-En-Tec Diagnostics, Taastrup, Denmark). Negative controls included omission of either the primary or secondary antibody and gave no signals (not shown). To detect degenerating neurons, Fluoro Jade B (Millipore AG310) was used. The 10-μm thin brain cryosections mounted on slides were immersed in a solution containing 1% sodium hydroxide in 80% alcohol followed by 70% alcohol and water. They were then transferred to a solution of 0.06% potassium permanganate for 10 minutes and rinsed in water. After 20 min staining with a concentration of 0.0004% Fluoro Jade B, the slides were rinsed with water and then dried at approximately 50°C.
2.2.3 Magnetic resonance imaging
Morphological changes and progression of brain damage induced by weight drop can be assessed by magnetic resonance imaging (MRI) . The representative T1- and T2-weighted MR-images in Figure 7, were obtained with a 3.0- Tesla magnetic resonance apparatus (Vision, Siemens) using a brain coil for rodents 1 day and 14 days after induction of injury. We detected a contusion in the ipsilateral hemisphere the 1st day after injury, and on the 14th day, enlarged ventricles were observed, indicating brain atrophy.
In this article we provide a thorough methodological description of a straightforward weight-drop mouse model reproducing brain injuries similar to those experienced in the human with a mix of diffuse injury pattern and focal contusion. A major advantage of this model is that it allows neurological scoring immediately after injury and in the sequel. Thus, although the inflicted brain injury varies in its severity, clinically relevant randomization of mice into the various treatment groups is possible and this animal model might facilitate the discovery of improved drug treatments for TBI.
This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding programme Open Access Publishing.
- Gaetz M: The neurophysiology of brain injury. Clin Neurophysiol 2004, 115: 4–18. 10.1016/S1388-2457(03)00258-XPubMedView ArticleGoogle Scholar
- Faul M, Xu L, Wald MM, Coronado VG: Traumatic Brain Injury in the United States: Emergency Department Visits, Hospitalizations, and Deaths, 2002–2006. In Book Traumatic Brain Injury in the United States: Emergency Department Visits, Hospitalizations, and Deaths, 2002–2006. City; 2010.Google Scholar
- Maegele M, Engel D, Bouillon B, Lefering R, Fach H, Raum M, Buchheister B, Schaefer U, Klug N, Neugebauer E: Incidence and outcome of traumatic brain injury in an urban area in Western Europe over 10 years. Eur Surg Res 2007, 39: 372–379.PubMedView ArticleGoogle Scholar
- Mock C, Joshipura M, Goosen J, Lormand JD, Maier R: Strengthening trauma systems globally: the Essential Trauma Care Project. J Trauma 2005, 59: 1243–1246.PubMedView ArticleGoogle Scholar
- Yattoo G, Tabish A: The profile of head injuries and traumatic brain injury deaths in Kashmir. J Trauma Manag Outcomes 2008, 2: 5. 10.1186/1752-2897-2-5PubMed CentralPubMedView ArticleGoogle Scholar
- Chiu WT, Huang SJ, Tsai SH, Lin JW, Tsai MD, Lin TJ, Huang WC: The impact of time, legislation, and geography on the epidemiology of traumatic brain injury. J Clin Neurosci 2007, 14: 930–935. 10.1016/j.jocn.2006.08.004PubMedView ArticleGoogle Scholar
- Martins ET, Linhares MN, Sousa DS, Schroeder HK, Meinerz J, Rigo LA, Bertotti MM, Gullo J, Hohl A, Dal-Pizzol F, Walz R: Mortality in severe traumatic brain injury: a multivariated analysis of 748 Brazilian patients from Florianopolis City. J Trauma 2009, 67: 85–90. 10.1097/TA.0b013e318187aceePubMedView ArticleGoogle Scholar
- Albert-Weissenberger C, Siren AL: Experimental traumatic brain injury. Exp Transl Stroke Med 2010, 2: 16. 10.1186/2040-7378-2-16PubMed CentralPubMedView ArticleGoogle Scholar
- Rakos G, Kis Z, Nagy D, Lur G, Farkas T, Hortobagyi T, Vecsei L, Toldi J: Evans Blue fluorescence permits the rapid visualization of non-intact cells in the perilesional rim of cold-injured rat brain. Acta Neurobiol Exp (Wars) 2007, 67: 149–154.Google Scholar
- Raslan F, Schwarz T, Meuth SG, Austinat M, Bader M, Renne T, Roosen K, Stoll G, Siren AL, Kleinschnitz C: Inhibition of bradykinin receptor B1 protects mice from focal brain injury by reducing blood-brain barrier leakage and inflammation. J Cereb Blood Flow Metab 2010, 30: 1477–1486. 10.1038/jcbfm.2010.28PubMed CentralPubMedView ArticleGoogle Scholar
- Stoffel M, Blau C, Reinl H, Breidt J, Gersonde K, Baethmann A, Plesnila N: Identification of brain tissue necrosis by MRI: validation by histomorphometry. J Neurotrauma 2004, 21: 733–740. 10.1089/0897715041269678PubMedView ArticleGoogle Scholar
- Eriskat J, Furst M, Stoffel M, Baethmann A: Correlation of lesion volume and brain swelling from a focal brain trauma. Acta Neurochir Suppl 2003, 86: 265–266. 10.1007/978-3-7091-0651-8_57PubMedGoogle Scholar
- Siren AL, Radyushkin K, Boretius S, Kammer D, Riechers CC, Natt O, Sargin D, Watanabe T, Sperling S, Michaelis T, et al.: Global brain atrophy after unilateral parietal lesion and its prevention by erythropoietin. Brain 2006, 129: 480–489.PubMedView ArticleGoogle Scholar
- Flierl MA, Stahel PF, Beauchamp KM, Morgan SJ, Smith WR, Shohami E: Mouse closed head injury model induced by a weight-drop device. Nat Protoc 2009, 4: 1328–1337. 10.1038/nprot.2009.148PubMedView ArticleGoogle Scholar
- Foda MA, Marmarou A: A new model of diffuse brain injury in rats. Part II: Morphological characterization. J Neurosurg 1994, 80: 301–313. 10.3171/jns.1994.80.2.0301PubMedView ArticleGoogle Scholar
- Marmarou A, Foda MA, van den Brink W, Campbell J, Kita H, Demetriadou K: A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics. J Neurosurg 1994, 80: 291–300. 10.3171/jns.1994.80.2.0291PubMedView ArticleGoogle Scholar
- Loane DJ, Pocivavsek A, Moussa CE, Thompson R, Matsuoka Y, Faden AI, Rebeck GW, Burns MP: Amyloid precursor protein secretases as therapeutic targets for traumatic brain injury. Nat Med 2009, 15: 377–379. 10.1038/nm.1940PubMed CentralPubMedView ArticleGoogle Scholar
- Feeney DM, Boyeson MG, Linn RT, Murray HM, Dail WG: Responses to cortical injury: I. Methodology and local effects of contusions in the rat. Brain Res 1981, 211: 67–77. 10.1016/0006-8993(81)90067-6PubMedView ArticleGoogle Scholar
- Dail WG, Feeney DM, Murray HM, Linn RT, Boyeson MG: Responses to cortical injury: II. Widespread depression of the activity of an enzyme in cortex remote from a focal injury. Brain Res 1981, 211: 79–89. 10.1016/0006-8993(81)90068-8PubMedView ArticleGoogle Scholar
- Panikashvili D, Simeonidou C, Ben-Shabat S, Hanus L, Breuer A, Mechoulam R, Shohami E: An endogenous cannabinoid (2-AG) is neuroprotective after brain injury. Nature 2001, 413: 527–531. 10.1038/35097089PubMedView ArticleGoogle Scholar
- Chen Y, Constantini S, Trembovler V, Weinstock M, Shohami E: An experimental model of closed head injury in mice: pathophysiology, histopathology, and cognitive deficits. J Neurotrauma 1996, 13: 557–568.PubMedView ArticleGoogle Scholar
- Shapira Y, Shohami E: Experimental studies on brain oedema after blunt head injury: experimental approaches from animal experimentation to actual or possible clinical application. Eur J Anaesthesiol 1993, 10: 155–173.PubMedGoogle Scholar
- Shukla D, Devi BI, Agrawal A: Outcome measures for traumatic brain injury. Clinical Neurology and Neurosurgery 2011, 113: 435–441. 10.1016/j.clineuro.2011.02.013PubMedView ArticleGoogle Scholar
- Castejon OJ: Morphological astrocytic changes in complicated human brain trauma. A light and electron microscopic study. Brain Inj 1998, 12: 409–427. discussion 407 10.1080/026990598122539PubMedView ArticleGoogle Scholar
- Castejon OJ: Electron microscopic analysis of cortical biopsies in patients with traumatic brain injuries and dysfunction of neurobehavioural system. J Submicrosc Cytol Pathol 1998, 30: 145–156.PubMedGoogle Scholar
- Oehmichen M, Meissner C: Routine techniques in forensic neuropathology as demonstrated by gunshot injury to the head. Legal Medicine 2009,11(Supplement 1):S50-S53.PubMedView ArticleGoogle Scholar
- Oehmichen M, Walter T, Meissner C, Friedrich HJ: Time course of cortical hemorrhages after closed traumatic brain injury: statistical analysis of posttraumatic histomorphological alterations. J Neurotrauma 2003, 20: 87–103. 10.1089/08977150360517218PubMedView ArticleGoogle Scholar
- Kleinschnitz C, Stoll G, Bendszus M, Schuh K, Pauer HU, Burfeind P, Renne C, Gailani D, Nieswandt B, Renne T: Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis. J Exp Med 2006, 203: 513–518. 10.1084/jem.20052458PubMed CentralPubMedView ArticleGoogle Scholar
- Tsenter J, Beni-Adani L, Assaf Y, Alexandrovich AG, Trembovler V, Shohami E: Dynamic changes in the recovery after traumatic brain injury in mice: effect of injury severity on T2-weighted MRI abnormalities, and motor and cognitive functions. J Neurotrauma 2008, 25: 324–333. 10.1089/neu.2007.0452PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.