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 bodies⁴. These aggregates primarily consist of alpha-synuclein (asyn), a small but highly expressed protein in neurons in the brain⁵.

The underlying mechanism of neurodegeneration in PD is still unknown. Aging is still the biggest risk factor for this disorder⁶. Furthermore, humans are the only species that develops PD naturally. Therefore, in order to investigate PD pathology and test new drugs to prevent disease progression, a wide array of animal models have been developed⁷. Ideally, animal models of PD should display an age dependent, progressive loss of DA neurons in the SN, accompanied by intracellular inclusions followed by motor dysfunction and be responsive to DA replacement therapies. None of the currently available animal models fully recapitulate all clinical symptoms and pathology of PD. As each model presents with different aspects of the disease, it is important to carefully consider the appropriate model to use in an experiment based on the questions asked.

Historically, animal models were based on toxicants, including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and pesticides, such as rotenone and paraquat⁸. Each toxicant has a different mechanism of action and ranges from DA neuron specific to generally harmful to brain cells. Toxins can either be given orally, injected intraperitoneally or directly into the brain using stereotaxic injections depending on blood brain barrier permeability. Unlike other models, toxin models guarantee a high degree of nigrostriatal dopaminergic cell loss and behavioral phenotypes. Some models may even present with subtle pathology. These features make toxin PD models a great tool for studying replacement therapies and the effects of environmental toxins on the onset of PD^9,10.

Additionally, numerous transgenic mouse models have been generated using a variety of promoters and PD related genes¹¹. Most mice present with nigrostriatal pathology but without clear evidence of neurodegeneration. Transgenic models have the advantage of being consistent between animals and cohorts and once generated are easy to maintain and distribute. While they do not result in neurodegeneration, they are nevertheless useful models to investigate cellular changes caused by genetic variants and possible drug candidates in a complex in vivo system¹².

In contrast to transgenic models, viral vector mediated expression of PD related genes offers a more flexible approach¹³. Stereotaxic injections allow for various brain areas, cell types, and expression levels to be chosen for a broad range of animal species such as mice, rats, pigs and non-human primates. Initially, recombinant viral vectors encoding for asyn were used to transduce neurons located in the rat SN. Protein accumulation and cellular dysfunction precede progressive dopaminergic cell loss resulting in behavioral deficit. Differences in targeting can lead to a large variation of cell loss between animals (30-80%), which is responsible for variable behavioral deficits seen in only approximately 25% of injected rats¹⁴.

A recently established model is the intracranial injection of preformed asyn fibrils (PFFs) or aggregate extracts from mouse or patient brain tissue^15,16. Multiple studies indicate that the injection of PFFs or extracts result in a wide-spread asyn pathology in the animal brain as well as a loss of dopaminergic neurons in the SN. Accumulation of asyn appears within neurons innervating the injected area. Unlike viral vector-based models, the PFF model develops slowly over several months followed by motor deficits at 6 months. This model has great potentialfor studying the mechanism or prevention of asyn pathology^17,18.

All models mentioned above have been well-established and used numerous times to study various aspects of the human disorder. Stereotaxic injections of substances directly into the brain have played a large part in the development of these animal models not only in the field of PD but also other neurological disorders. While it is labor-intensive, stereotaxic surgery has the advantages of being highly flexible in age of animals used, brain region targeted and substance injected, and can be adjusted depending on the research question asked. For example, substances can be injected singly or in combination (vector + fibrils or toxicant + vector) to recapitulate more aspects of the disease or assess treatments^19,20. Additionally, substances can be injected unilaterally leaving the uninjected side as an internal control for evaluating behavior as well as neurodegeneration. Therefore, this manuscript will outline detailed steps to generate PD models using stereotaxic injections.

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 fentan…

Representative 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…

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

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