Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion
Summary
This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.
Abstract
Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.
Introduction
Mild TBI, including concussion and sub-concussion, account for the majority of all TBI cases (>80% of all TBI)1. Mild TBI commonly results from falls, traffic accidents, acts of violence, contact sports (e.g., football, boxing, hockey), and military combat2,3. Mild TBI can lead to neurobiological events that affect neurobehavioral functions throughout the patient's lifetime and increase the risk of neurodegenerative diseases4,5,6. Animal models provide an efficient and controlled means to study mild TBI, with the hope of further enhancing the diagnosis and treatment of mild TBI. Various models for mild TBI have been developed, such as the controlled cortical impact (CCI), weight drop (WD), fluid percussion injury (FPI), and blast-TBI models7,8. No single experimental model can mimic the entire complexity of TBI-induced pathology9,10. The heterogeneity of these models is advantageous for addressing the diverse features associated with mild TBI patients and investigating the corresponding cellular and molecular mechanisms. However, each animal model of TBI has its limitations3, limiting our current knowledge concerning animal mild TBI and their clinical relevance.
The WD and CCI models are utilized to replicate clinical conditions such as cerebral tissue loss, acute subdural hematoma, axonal injury, brain concussion, blood-brain barrier dysfunction, and even coma following TBI3,11,12. The WD model involves inducing brain damage by striking either the dura mater or skull with freely falling weights. The impact of a weighted object upon an intact skull can replicate mixed focal/diffuse injuries; however, this method is associated with poor accuracy and repeatability of the injury site, rebound injury, and a higher mortality rate due to skull fractures3,11,12. The CCI model involves applying air-driven metal to impact the exposed dura mater directly. Compared to the WD model, the CCI model is more accurate and reproducible, but it does not produce diffuse injury due to the small diameter of the impacting tip11. During FPI modelling, the brain tissue is briefly displaced and deformed by percussion. FPI can induce mixed focal/diffuse injury and replicate intracranial hemorrhage, brain swelling and progressive gray matter damage after TBI. However, FPI has a high mortality rate due to brainstem damage and prolonged apnea3,12. The craniotomy involved in conventional WD, CCI, and FPI models can lead to cortical contusion, hemorrhagic lesions, the damage of the blood-brain barrier, immune cell infiltration, glial cell activation, prolonged modeling time, and possible fatal outcomes3,12.
Mild TBI is characterized by a GCS (Glasgow coma scale, GCS) score within the range of 13 to 152. Mild TBI can be either focal or diffuse and is associated with both acute injuries, such as breakdown of cellular homeostasis, excitotoxicity, glucose depletion, mitochondrial dysfunction, blood flow disturbance, and axonal damage, as well as subacute injuries, including axonal damage, neuroinflammation, and gliosis2,3. Despite significant progress in delineating the intricate pathophysiology of TBI, the underlying mechanisms of mild TBI/rmTBI remain elusive and require further investigation9. Given that CHI is the most common type of TBI12, this protocol presents a novel approach to creating a more precisely controlled mouse model of rmTBI using a modified FPI device to perform impact in a thinned-skull window13. By avoiding craniotomy-induced injuries, variable skull thickness and shape-induced inaccuracies, and rebound injury, this approach aims to overcome the main disadvantages associated with the WD, CCI, and FPI models. Applying FPI impact on the thinned-skull window is convenient for evaluating cerebral vessel damage following rmTBI and helps minimize high mortality rates in some models, resulting in a closer resemblance to the clinical features of TBI patients.
Protocol
Representative Results
Discussion
TBI refers to two primary types, closed and penetrating, with the latter characterized by a disruption of the skull and dura mater. Clinical data suggest that CHIs are more prevalent than penetrating injuries1,2. After a single mild TBI, most patients experience PCS symptoms that typically resolve in a short period of time, and there is controversy regarding the proportion of patients whose PCS develop into long-term sequelae23,…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Key Social Development Foundation of Jinhua Municipality (No. 2020-3-071), Zhejiang College Student Innovation and Entrepreneurship Training Program (No: S202310345087, S202310345088) and Zhejiang Provincial College Students' Science and Technology Innovation Activity Plan Project (2023R404044). The authors thank Miss Emma Ouyang (first-year student of Johns Hopkins University, Bachelor of Science, Baltimore, USA) for language editing the article.
Materials
| 75% ethanol | Shandong XieKang Medical Technology Co., Ltd. | 220502 | |
| Buprenorphine hydrochloride | Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd | H12020272 | Solution, Analgesic |
| Carprofen | Shanghai Guchen Biotechnology Co., Ltd | 53716-49-7 | Powder, Analgesic |
| Chlorhexidine digluconate | Shanghai Macklin Biochemical Co.,Ltd. | 18472-51-0 | 19%-21% aqueous solution, Antimicrobial |
| Dental cement and solvent kit | Shanghai New Century Dental Materials Co., Ltd. | 20220405, 3# | Powder reconsituted in matching solvent |
| Dissecting microscope | Shenzhen RWD Life Science Inc. | 77019 | |
| Erythromycin ointment | Wuhan Mayinglong Pharmaceutical Group Co.,Ltd. | 220412 | Antibiotic |
| Fiber Optic Cold Light Source | Shenzhen RWD Life Science Inc. | F-150C | |
| Flat-tipped micro-drill bit | Shenzhen RWD Life Science Inc. | HM31008 | 2 mm, steel |
| FPI device software | Jiaxing Bocom Biotech Inc. | Biocom Animal Brain Impactor V1.0 | |
| ICR mice | Jinhua Laboratory Animal Center | Stock#2023091 | 25 Male mice, 25-30g, 8 weeks old |
| Isoflurane | Shandong Ante Animal Husbandry Technology Co., Ltd. | 2023090501 | |
| Isothermal heating pad | Wenzhou Repshop Pet Products Co., Ltd. | ||
| Luer Loc hup | Custom made using a 19G needle hub | ||
| Micro hand-held skull drill | Shenzhen RWD Life Science Inc. | 78001 | Max: 38,000rpm |
| Modified FPI device | Jiaxing Bocom Biotech Inc. | ||
| Morris water maze | Shenzhen RWD Life Science Inc. | 63031 | Evaluate mouse spatial learning and memory abilities |
| Open field | Shenzhen RWD Life Science Inc. | 63008 | Evaluate mouse locomoation and anxiety |
| Ophthalmic lubricant | Suzhou Tianlong Pharmaceutical Co., Ltd. | SC230724B | |
| Sodium diclofenac ointment | Wuhan Mayinglong Pharmaceutical Group Co.,Ltd. | 221207 | nonsteroidal anti-inflammatory drug |
| Small animal anesthesia system-Enhanced | Shenzhen RWD Life Science Inc. | R530IP | |
| Smart video-tracking system | Panlab Harvard Apparatus Inc., MA, USA | V3.0 | Animal tracking and analysis |
| Stereotactic frame | Shenzhen RWD Life Science Inc. | 68043 | |
| Vetbond Tissue Adhesive | 3M, St Paul, MN, USA | 202402AX | Suture the animal wound |
| Y maze | Shenzhen RWD Life Science Inc. | 63005 | Evaluate mouse spatial working memory |
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