Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model

Summary

The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking software.

Abstract

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson's disease (PD) raise the need for alternative therapies that can restore these neurons. Much effort is currently directed toward a better understanding of neuroregeneration using preclinical in vivo models. This regenerative capability for self-repair is, however, inefficient in mammals. Non-mammalian animals like zebrafish have thus emerged as an excellent neuroregenerative model due to its capability to continuously self-renew and have a close brain homology to humans. As part of the effort in elucidating cellular events involved in neuroregeneration in vivo, we have established the 6-hydroxydopamine (6-OHDA)-induced adult zebrafish-based PD model. This was achieved through the optimized intracerebroventricular (ICV) microinjection of 99.96 mM 6-OHDA to specifically ablate dopaminergic neurons (DpN) in the ventral diencephalon (Dn) of zebrafish brain. Immunofluorescence indicated more than 85% of DpN ablation at day three postlesion and full restoration of DpN at lesioned site 30 days postlesion. The present study determined the impairment and subsequent recovery of zebrafish swimming behavior following lesion by using the open field test through which two parameters, distance traveled (cm) and mean speed (cm/s), were quantified. The locomotion was assessed by analyzing the recordings of individual fish of each group (n = 6) using video tracking software. The findings showed a significant (p < 0.0001) reduction in speed (cm/s) and distance traveled (cm) of lesioned zebrafish 3 days postlesion when compared to sham. The lesioned zebrafish exhibited full recovery of swimming behavior 30 days postlesion. The present findings suggest that 6-OHDA lesioned adult zebrafish is an excellent model with reproducible quality to facilitate the study of neuroregeneration in PD. Future studies on the mechanisms underlying neuroregeneration as well as intrinsic and extrinsic factors that modulate the process may provide important insight into new cell replacement treatment strategies against PD.

Introduction

Parkinson's disease (PD), a disease distinctively characterized by muscle rigidity, resting tremor, and bradykinesia, is the fastest growing neurological disease in the world^1,2. The risk and prevalence of PD increase rapidly with age especially in individuals aged 50 years and above³. The etiology and pathogenesis of PD hitherto remain poorly understood. This has often left the early-onset of PD undiagnosed. At present, the lack of dopamine and the loss of dopaminergic neurons (DpN) in PD patients are strongly linked to the manifestation of motor symptoms⁴. ....

Protocol

The present study has been approved by the Committee on Animal Research and Ethics (CARE), Universiti Technologi MARA (UiTM) [Reference No: UiTM CARE 346/2021, dated 7 May 2021].

NOTE: The published protocols^22,25,26 for standard husbandry and maintenance of the 6-OHDA-lesioned adult zebrafish PD model were utilized. Experiments were conducted with adult male zebrafish (Danio rerio) aged more than five months old with a standardized length of 3.2-3.7 cm.

1. Zebrafish maintenance and pre-ICV microinjection ....

Results

The present experiment assessed the changes in adult zebrafish swimming behavior following ICV microinjection with 6-OHDA. The reason for using 6-OHDA as the neurotoxin of choice was due to its inability to cross the blood-brain barrier, which produced specific and targeted ablation of DpN in the area of interest-ventral diencephalon (Dn)¹⁶. The DpN subpopulation here holds anatomical resemblance to the DpN subpopulation in the human's substantia nigra pars compacta³¹.<.......

Discussion

The present work successfully demonstrated the locomotor assessment of the established 6-OHDA-induced, adult zebrafish-based PD model. The entire experiment involved three major steps: pre-ICV microinjection preparations, ICV microinjection of zebrafish, and locomotor assessment. To ensure the healthy recovery of adult zebrafish following the ICV microinjection procedure and good experimental outcome, some good practices for each step have been recommended in the present study.

Pre-ICV microin.......

Materials

Name	Company	Catalog Number	Comments
Materials
6-Hydroxydopamine (6-OHDA)	Sigma-Aldrich, Missouri, USA	162957
Ascorbic acid	Thermo Fisher Scientific, California, USA	FKC#A/8882/53
Disposable pasteur pipette, 3 mL	Thermo Fisher Scientific, California, USA	FB55348
Microcentrifuge tube, 0.2 mL	Eppendorf, Hamburg, Germany	30124332
Nice conical flask, 100 mL	Evergreen Engineering & Resources, Semenyih, Malaysia	SumYau0200
Phosphate buffered saline (PBS)	Sigma-Aldrich, Missouri, USA	P4417
Sodium bicarbonate	Sigma-Aldrich, Missouri, USA	S5761
Sodium chloride	Merck, Darmstadt, Germany	106404
Stereomicroscope	Nikon, Tokyo, Japan	SMZ745
Tricaine methanesulfonate (MS-222)	Sigma-Aldrich, Missouri, USA	E10521
Equipment
ANY-maze software	Stoelting Co., Illinois, USA	-	version 7.0; video tracking software
Cubis II Micro Lab Balance	Sartorius, Göttingen, Germany	SE 2
FemtoJet IV microinjector	Eppendorf, Hamburg, Germany	5192000035
Femtotip II, sterile injection capillary	Eppendorf, Hamburg, Germany	5242957000
InjectMan 4 micromanipulator	Eppendorf, Hamburg, Germany	5192000027
LED Portable Lamp	MR. DIY, Selangor, Malaysia	9023251	20 mAh
PELCO Pro Superalloy, offset, fine tips	Ted Pella, California, USA	5367-12NM
Shanda aquarium heater	Yek Fong Aquarium, Selangor, Malaysia	SDH-228
Thermometer	Sera Precision, Heinsberg, Germany	52525
Video camera	Nikon, Tokyo, Japan	D3100