Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease

Summary

We describe how to successfully inject solutions into specific brain areas of rodents using a stereotaxic frame. This survival surgery is a well-established method used to mimic various aspects of Parkinson's disease.

Abstract

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

Introduction

Parkinson's disease (PD) is a relatively common progressive neurodegenerative disease affecting up to 1 % of people over the age of 60¹. PD is heterogenousbut clinically characterized mainly by motor symptoms including resting tremor, bradykinesia, akinesia, rigidity, gait disturbance and postural instability. The majority of motor symptoms typically appear when 60-70% of striatal dopamine (DA) is lost as a result of progressive and distinct neurodegeneration in the substantia nigra (SN) pars compacta^2,3. Surviving dopaminergic neurons contain intracellular inclusions known as Lewy ....

Protocol

All experiments in this study were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Animal Care and Use Committees of the US National Institute on Aging.

Before starting, please make sure to have acquired the appropriate training and ethical approval from your institute necessary to perform this procedure. Additionally, anesthetics (e.g., ketamine and buprenorphine, or fentanyl and medetomidine) used should be acquired and handled according to relevant rules of your institution.

1. Preparation (du....

Results

To avoid mistargeting, before every experiment, verify the coordinates using dye injections. Animals were injected with 0.2-0.5 µL tryptophan blue using the same protocol, capillary was rapidly withdrawn after injection and the brain was quickly frozen to avoid diffusion. After sectioning on the microtome, the injection site can be seen in blue (Figure 2 C,E). To ensure effective targeting, dye injections should be carried out successfully on 2-3 animals prior to actual expe.......

Discussion

Stereotaxic injection, as any surgical procedure, has the main difficulty to guarantee the wellbeing and survival of the animal. Therefore, it is essential to monitor the animal closely throughout the procedure. Looking out for breathing irregularities, loss of breathing, or reoccurrence of reflexes and movements should be the main focus, especially for inexperienced surgeons. Additionally, the application of analgesics is crucial to help with the recovery process. Surgeries involving toxicants can be especially difficul.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Allen brain atlas	Allen Institute	mouse brain - reference atlas
analgesic: ketoprofin OR buprenorphine
anesthetic: Isoflurane OR ketamine / xylazine OR fentanyl / medetomidine
blades - surgical sterile	Oasis Medical	No 10
capillaries - glass	Stoelting	50811
capillary puller	Sutter Instruments	P-97
cotton-tipped applicators	Stoelting	50975
drill - dental	Foredom	MH-170
Ethanol 70%
eye drops (Liquigel)	CVS	NDC 0023-9205-02	Carboxymethylcellulose Sodium (1%), Boric acid; calcium chloride; magnesium chloride; potassium chloride; purified water; PURITE® (stabilized oxychloro complex); sodium borate; and sodium chloride
forceps - full curved	Stoelting	52102-38P
forceps - hemostatic delicate	Stoelting	52110-13
gauze - cotton absorbent
H2O - sterile
H2O2 30%	Sigma Aldrich	216763
Hamilton 5ul syringe	Hamilton Company	7634-01
Hamilton blunt metal needle	Hamilton Company	7770-01
heat pad - far infrared	Kent Scientific	2665967
Iodine solution (Dynarex) 10%	Indemedical	102538
isoflurane	Baxter	1001936040
lidocaine 0.5%
lighter / matches
microscope (Stemi 508 Boom stand)	Zeiss	435064-9000-000
PBS sterile	Gibco - Thermo Fischer	10010-023
pump (injector)	Stoelting	53311
scalpel handle	Stoelting	52171P
shaver - electrical	andis	64800
solution to inject / material to implant
stereotax - small animal digital	Kopf	Model 940
sterilizer - glass bead	BT Lab Systems	BT1703
tubing - heat-shrink	Nelco	NP221-3/64
tweezers - dumont fine curved	Roboz	RS-5045A
underpad - absorbent
vaporizer for isoflurane (package)	Scivena Scientific	M3000
wound clips and applier / remover	Stoelting	59040
wound glue (Vetbond)	3M corporation	1469SB