Roman Modell Mild traumatische Hirnverletzung für jungen Ratten
Summary
The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.
Abstract
Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.
Introduction
Obwohl es viele weit verbreitete Methoden zur Erzeugung von mittelschwerer bis schwerer traumatischer Hirnverletzung (TBI), sehr wenige Techniken entwickelt, um zu induzieren mild, geschlossene Kopfverletzungen bei Nagern. Aufgrund der Tatsache, dass die milden traumatischen Hirnverletzungen (mTBI) ist dreimal häufiger als mittelschweren und schweren Hirnverletzungen in Verbindung 1 ist ein zuverlässiges Modell der mTBI benötigt, um Forschung in Bezug auf Pathophysiologie neurobiologischen und Verhaltensergebnisse und therapeutische Strategien zu erleichtern. Zum Beispiel, teilweise aufgrund der Einschränkungen der aktuellen Tiermodellen 2, in den letzten zehn Jahren gab es mehr als 200 gewesen gescheitert klinischen Arzneimittelstudien für die Behandlung von TBI 3. Bei der Modellierung von Systemen für translationale Forschung Studien erstellt, die Anwendbarkeit der Ergebnisse sind abhängig von der Gültigkeit des Modells implementiert. Für die Studie der mTBI / Gehirnerschütterung, wäre eine zuverlässige Tiermodell nicht nur imitieren die biomechanischen Kräfte Verantwortlich für Verletzungen Ätiologie, aber auch Symptome, die mit den von der klinisch relevanten Bevölkerung berichtet induzieren. Darüber hinaus, weil Kinder ein besonders hohes Risiko für mTBI würde optimale Modellierungssysteme für junge und jugendliche Nagetiere, zusätzlich zu ihrem Erwachsenenäquivalente.
Biomechanische Analysen der Umstände, in denen Athleten mTBIs oder erschütternder Hirnverletzungen erlitten zeigen, dass die wichtigsten Prognosefaktoren für Verletzungen sind schnelle Kopfbeschleunigung und hoher Geschwindigkeit Auswirkungen 4. Die Mehrheit der Nagetiermodellen derzeit für die Induktion von TBI beschäftigt erlauben wenig oder keine Bewegung des Kopfes 5 (für einen Überblick siehe 2). Die hier vorgestellte Modell liefert ein Hochgeschwindigkeits-Aufprall des Kopfes eines physikalisch ungebremst juvenilen Ratten, die durch eine Drehung um 180 ° und im freien Fall, die Beschleunigungs- / Verzögerungskräften gilt für Kopf und Körper der Person begleitet wird. THier sind vor allem zwei Vorteile mit dieser modifizierten Gewicht Drop-Technik für die Induktion von mTBI verbunden. Zuerst das Modell produziert klinisch relevanten concussive wie Symptomatik, ohne dass eine offensichtliche Schädigung des Gehirns (für eine vollständige Beschreibung der Verhaltensergebnisse siehe 6). Auch im Einklang mit klinischen Berichte über post concussive Syndrom, diese modifizierte Gewicht-Drop-Technik produziert heterogene Ergebnisse. Obwohl die Auswirkungen der mTBI signifikant sind, gibt es erhebliche Unterschiede zwischen Nagern, die eine mTBI erlebt, wenn auf mehreren Zielparameter untersucht. Zweitens gestattet das Verfahren die Untersuchung der repetitiven mTBI 7. Da die Mehrheit der bestehenden TBI Modelle wie schwere Verletzungen verursachen, ist es oft schwierig, eine zweite Verletzung zu induzieren, und fast unmöglich, sich wiederholende TBI ohne großen Schaden für die gesamte Cortex studieren.
Daher ist die primäre Grund für die Verwendung der modifizierten Gewicht Drop-Technik für die Induction des mTBI ist es, eine Verletzung, die der Pathophysiologie und Symptomatik von Gehirnerschütterung und repetitive TBI in Jugendgruppen stärker repräsentiert zu produzieren. Mit der zunehmenden Häufigkeit von mTBI im Zusammenhang mit Sport, Stürze und Autounfälle, vor allem während der Kindheit, dieses einzigartige Nagetiermodell der mTBI bietet Forschern ein wertvolles Werkzeug für die Untersuchung von erschütternder artigen Hirnverletzung, die leicht mehrere Hit-angewendet werden können Paradigmen.
Protocol
Representative Results
Discussion
Reliable modelling systems are needed to effectively cultivate basic science research that has significant translational validity. In response to rising occurrences and popular media, the investigation of mTBI and concussion has become a priority in many disciplines. However, despite increased research, there have been only incremental improvements in therapeutic strategies and treatment options 3. This lack of progress may be partially due to a discrepancy between the modeling systems employed and actual injury etiology. The majority of studies utilized rodent models that failed to reproduce the important biomechanical forces and appropriate post-injury symptomology. The current human definition of mTBI specifies that the injury results from acceleration and deceleration forces associated with a blunt trauma 10. The modified weight drop technique described here is therefore an ideal model for the study of mTBI and concussion because it uses a glancing impact to cause rapid rotational acceleration and deceleration to the head of an unrestrained animal, mimicking the biomechanical forces identified in sports-related injuries and automobile accidents. In addition, this model is easily adapted to examine repetitive mTBI, a phenomena that is emerging as a serious medical and socioeconomic issue. Studies indicate that rodents may be exposed to a series of 10 distinct mTBIs with minimal mortality 7. Finally, the method is inexpensive and can be carried out rapidly, allowing for high-throughput examination of a many therapeutic compounds and treatment regiments.
Just as with any procedural technique, certain aspects of the protocol are particularly important to the generation of reliable results. First, the tin foil needs to be scored effectively. If the tin foil is not properly scored, the force imparted by the weight during the glancing impact will not be enough to propel the juvenile rat through the tin foil onto the collection sponge. In these situations, the rat will remain in the starting position (chest down on the tin foil) and the mTBI will result from the blunt trauma from the weight impacting the stationary head, not the rotational acceleration and deceleration desired. Second, during the induction of the mTBI and the sham injury, the level of anesthetic applied to each rat should be consistent. Owing to the fact that time-to-right is used as marker of mTBI, the researcher should try to ensure that animals receiving a mTBI and animals receiving a sham injury are exposed to similar levels of anesthetic. A major advantage to this technique over many other TBI procedures is the low level and duration of anesthesiology. However, the juvenile rat needs to be non-responsive to a toe or tail pinch to ensure they do not wake-up on the stage before the injury is induced. Finally, in order to maintain a consistent injury etiology, the positioning of the rat’s head is particularly important. Ideally the weight should impact the center of the dorsal surface of the head. Caution should be taken to avoid positioning the path of the weight too near the caudal/posterior portion of the head, as impacting the brainstem and cerebellum is associated with increased mortality and seizure activity.
Based upon the biomechanical pathophysiology of injury induction and the behavioural outcomes examined, the modified weight-drop technique appears to be a reliable model for the investigation of paediatric mTBI and concussion. Although preliminary studies of this novel model have assessed some basic molecular and structural changes 7 future studies will be needed to ascertain how the brain responds to a mTBI with this injury etiology. An in-depth analysis of the neuroanatomical and biological changes that occur at the cellular and epigenetic level would increase model validity and translational applicability. In addition to stimulating the generation of targeted pharmacological therapies, understanding the pathophysiological changes that occur in the brain in response to mTBI and concussion would also direct the research related to clinical biomarkers that have the ability to predict outcomes following injury.
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Irene Ma, Rose Tobais, and Jong Rho for their technical assistance. Funding was provided to MJE by the Department of Pediatrics at the University of Calgary, the Alberta Children’s Hospital Foundation (ACHF) and the Alberta Children’s Hospital Research Institute (ACHRI). The Postdoctoral fellowship for RM was provided by ACHF.
Materials
| Brass Weights | Ginsberg Scientific | 7-2500-2 | Need to have metal loop attached to base |
| Alluminum Foil | Alcan | Available at most grocery stores | |
| Masking Tape | Commercially available | ||
| U-Shaped Plastic Stand | Constructed by Laboratory | ||
| Clamp Stand | Sigma-Aldrich | Z190357 | |
| Plastic Guide Tube | Could be constructed or purchased at a hardware store | ||
| Fishing Line | Angler 10lb | Purchased from a sporting goods retailer | |
| Isoflurane | Pharmaceutical Partners of Canada | DIN 02237518 | Inhalation Anesthetic |
| Topical Lidocaine (30ml) | Astra Zeneca | DIN 0001694 | Xylocaine Jelly 2% |
| Cotton Swabs | Commercially available | ||
| Heating Pad – 3 heat setting | Commercially available | ||
| Stop Watch | Sportline | L303 | Purchased from a sporting goods retailer |
| Video Camera | Sony | HDR-CX260V | |
| Sprague Dawley Rats | Charles River Laboratories | SAS SD 40 | Male and females ordered from Charles River Laboratories and pups bred in-house |
| Balance Beam | Constructed by Laboratory |
Referencias
- DeWitt, D., Perez-Polo, R., Hulsebosch, C., Dash, P., Robertson, C. Challenges in the development of rodent models of mild traumatic brain injury. Journal of Neurotrauma. 30, 688-701 (2013).
- Xiong, Y., Mahmood, A., Chopp, M. Animal models of traumatic brain injury. Nature Reviews Neuroscience. 14, 128-142 (2013).
- Zhang, Z., Lerner, S., Kobiessy, F., Hayes, R., Wang, K., Yan, Q. Systems biology and the anostic approach to drug discovery and development to treat traumatic brain injury. Systems Biology in Drug Discovery and Development: Methods and Protocols, Methods in Molecular Biology. , .
- Viano, D., Casson, I., Pellmen, E. Concussion in profession football: Biomechanics of the struck player – Part 14. Neurosurgery. 61, 313-327 (2007).
- Marmarou, A., et al. A new model of diffuse brain injury in rats: Part 1 – Pathophysiology and biomechanics. Journal of Neurosurgery. 80, 291-300 (1994).
- Mychasiuk, R., Farran, A., Esser, M. J. Assessment of an experimental rodent model of pediatric mild traumatic brain injury. Journal of Neurotrauma. 31 (8), (2014).
- Kane, M., et al. A mouse model of human repetitive mild traumatic brain injury. Journal of Neuroscience Methods. 203, 41-49 (2012).
- Schallert, T., Woodlee, M., Fleming, S., Krieglstein, J., Klumpp, S. Disentangling multiple types of recovery from brain injury. Pharmacology of Cerebral Ischemia. , (2002).
- Mychasiuk, R., Hehar, H., Farran, A., Esser, M. J. Mean Girls: Sex differences in the effects of mild traumatic brain injury on the social dynamics of juvenile rat play behaviour. Behavioural Brain Research. 259, (2014).
- Centers for Disease Control and Prevention. . Report to congress on mild traumatic brain injury in the United States: Steps to prevent a serious public health problem. , 1-47 (2003).
