A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Summary

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

Abstract

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

Introduction

Traumatic brain injuries (TBIs) are a global health crisis and a leading cause of death and disability. In the United States, approximately 2.9 million people experience a TBI each year, and between 2006-2014 mortality due to TBI or TBI sequelae increased by over 50%¹. However, TBIs vary in their etiology, pathology, and clinical presentation due largely in part to the mechanism of injury (MOI), which also influences treatment strategies and predicted prognosis². Though TBIs can result from various MOI, they are predominately the result of either a penetrating or blunt-force trauma. Penetrating traumas represent a small ....

Protocol

Zebrafish were raised and maintained in the Notre Dame Zebrafish facility in the Freimann Life Sciences Center. The methods described in this manuscript were approved by the University of Notre Dame Animal Care and Use Committee.

1. Traumatic brain injury paradigm

1. Add 1 mL of 2-phenoxyethanol to 1 L of system water (60 mg of Instant Ocean in 1 L of deionized RO water).
2. Prepare an aerated recovery tank containing 2 L of system water at room temperature.
3. Select the desired weight of ball bearing and a desired length and diameter of steel/plastic tubing and determine the energy and impact force.
   NOTE: Tub....

Results

Preparing the injury-induction rig allows for a rapid and simplistic means of delivering a scalable blunt-force TBI to adult zebrafish. The graded severity of the injury model provides several easily identifiable metrics of successful injury, though the vascular injury is one of the easiest and most prominent pathologies (Figure 3). The strain of fish used during the injury can make this indicator easier or harder to identify. When using wild-type AB fish(WT^AB, <.......

Discussion

Investigations of neurotrauma and associated sequelae have long been centered on traditional non-regenerative rodent models²⁰. Only recently have studies applied various forms of CNS damage to regenerative models^{9,11,13,14,21}. Though insightful, these models are limited by either their use of an injury method uncommonly seen in the huma.......

Materials

Name	Company	Catalog Number	Comments
2-phenoxyethanol	Sigma Alderich	77699
#00 buckshot	Remington	RMS23770	3.3g weight for sTBI
#3 buckshot	Remington	RMS23776	1.5g weight for miTBI/moTBI
#5 Dumont forceps	WPI	14098
5-ethynyl-2’-deoxyuridine	Life Technologies	A10044	EdU
5ml glass vial	VWR	66011-063
Click-iT EdU Cell Proliferation Kit	Life Technologies	C10340
CytoOne 12-well plate	USA Scientific	CC7682-7512
Instant Ocean	Instant Ocean	SS15-10
Super frost postiviely charged slides	VWR	48311-703
Super PAP Pen Liquid Blocker	Ted Pella	22309
Tissue freezing medium	VWR	15148-031