少年ラット用マイルド外傷性脳損傷の新規モデル
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
中等度から重度の外傷性脳損傷（TBI）の生成のための多くの広く使用されている方法があるが、非常に少数の技術は穏やかな誘導するために開発されており、げっ歯類における頭部外傷を閉じた。軽度の外傷性脳損傷（MTBI）は1を組み合わせた中等度および重度の脳損傷の3倍一般的であるという事実のために、MTBIの信頼性モデルは、病態生理、神経生物学および行動の結果、および治療 戦略に関する研究を促進するために必要とされる。たとえば、200の上にありました、過去10年間で、現在の動物モデル2の制限などの理由では、TBI 3の治療のための臨床薬物試験を失敗した。モデリングシステムはトランスレーショナルリサーチ研究のために生成される場合、調査結果の適用が実装モデルの妥当性に依存している。 MTBI /脳震盪の研究のために、信頼性の高い動物モデルは、生体力学的な力のresponを模倣していないだろう傷害の病因のためsibleするだけでなく、臨床的に関連する人口によって報告されたものと一致する症状を誘発するだろう。子供はMTBI特に高い危険にさらされているので、また、最適なモデリングシステムは、成人同等に加えて、若い少年げっ歯類に適用可能である。
選手がmTBIsまたは震とう脳損傷を負っているような状況の生体力学的分析は怪我のために最も重要な予測因子は、急速なヘッド加速と高速の影響4であることを示している。現在TBIの誘導のために用いられるげっ歯類モデルの大部分は、ヘッド5（総説については2を参照）のほとんど又は全く動きを可能にする。ここで概説モデルは、被験者の頭と体に加速/減速力を加える180°回転し、自由落下を伴う物理的に拘束されていない幼若ラットの頭部に高速のインパクトを実現します。 Tここでは、MTBIの誘導のため、この修正された重量損失の技術に関連する2つの主要な利点である。まず、モデルは（6を参照の行動の結果の完全な説明のために）は、脳への明白な損傷を与えることなく、症状のような震とう臨床的に関連を生成します。また、この修正された重量ドロップ技術が異質な成果をポスト震とう症候群の臨床報告と一致して生成します。 MTBIの影響が有意であるが、複数のアウトカム指標に検討したときにMTBIを経験したげっ歯類の間に実質的な変動がある。第二に、この方法は、反復MTBI 7の研究を可能にします。既存のTBIモデルの大部分は重篤な傷害を負わせるように、全体の皮質への広範な損傷を与えることなく、反復的なTBIを研究するために第二の損傷、およびほぼ不可能を誘導することがしばしば困難である。
したがって、寄り付きために改変重量ドロップ技術を使用する主な根拠MTBIのctionはより密接に脳震盪と少年集団における反復的なTBIの病態生理と症状を表してけがを生成することである。スポーツに関連するMTBIの発生率の増加により、落下し、自動車事故、特に小児期に、MTBIのこのユニークなげっ歯類モデルが簡単に複数のヒットに適用することができ震とうのような脳損傷の研究のための貴重なツールと研究者を提供していますパラダイム。
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.
Disclosures
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 |
References
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