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Neuroscience

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

Published: February 18th, 2021

DOI:

10.3791/62128

1Cell Biology and Gene Expression Section, National Institute on Aging, National Institutes of Health, 2Behavioral Neuroscience Program, Department of Psychology, Binghamton University, 3Department of Translational Neuroscience, Mercy Health Hauenstein Neuroscience Medical Center, Michigan State University

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.

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.

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 compacta2,3. Surviving dopaminergic neurons contain intracellular inclusions known as Lewy ....

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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 fentan.......

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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.......

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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.......

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This research was supported in part by the Intramural Research Program of the National Institute of Health, National Institute on Aging. CES is supported by NS099416. The authors wish to acknowledge support by the NIMH IRP Rodent Behavioral Core (ZIC MH002952 and MH002952 to Yogita Chudasama) and by the NICHD IRP Microscopy and Imaging Core.

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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

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