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 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 PD9,10.
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 tissue15,16. 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 pathology17,18.
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 treatments19,20. 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.
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 (duration 1 hour)
2. Surgery (duration average 1 hour per animal)
3. Post-OP care (duration 3-7 days)
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 experiment.
Stereotaxic injections can be used to create three main Parkinson's disease models with varying degrees of pathology and neurodegeneration. Typically, in the PD field, neurodegeneration is evaluated by quantifying tyrosine hydroxylase (TH), a marker for dopaminergic neurons, as well as a general neuronal marker (e.g., Neuronal Nuclei [NeuN] or HuC) as downregulation of TH due to a toxic environment can give the illusion of nigral cell death.
When injecting toxicants such as 6-OHDA the goal is to eradicate as many nigral dopaminergic cells as possible. 6-OHDA is taken up by monoamine transporters and blocks mitochondrial respiration21. Desipramine is given before each surgery to ensure the toxicant is only taken up by dopamine neurons and not by noradrenergic or serotonergic cells. To evaluate the success of each injection, fixed midbrain sections should be Immunolabeled for TH and NeuN. As toxins act quickly, this model only takes 2-3 weeks after injection to be fully developed. If 6-OHDA injections into the medial forebrain bundle (MFB) were successful, dopaminergic cell loss should be 80% and greater compared to PBS injected controls (Figure 2 F-G). The MFB is a region where nigrostriatal projections bundle together and therefore allow for targeting many dopaminergic neurons with a single injection. Toxicants can also be injected directly into the nigra or striatum for less severe cell death.
Viral vector-based models highly depend on the (sero-)type of the vector used. AAV2 was the most commonly injected as it was easy to produce and has a high affinity towards dopaminergic neurons. Engineering of expression promoters, viral capsids and envelope proteins has opened doors to more specific targeting of brain regions and cell populations. Most viral vectors in PD models are directly injected into the SN to transduce dopaminergic neurons in the pars compacta. Expression of most vectors stabilizes 3 weeks after injection and can be visualized by immunolabeling for the exogenous protein. Usually, GFP is expressed as a control at titers not causing neurodegeneration (Figure 2H). PD-related genes are used to mimic the genetic aspect of the disease, for example duplications or triplications of asyn. Overexpression of asyn in nigral neurons causes dopaminergic cell death (Figure 2I).
The PFF model is the newest addition to the PD tool kit. Aggregated asyn can be acquired either in vitro by weeklong shaking of recombinant asyn protein to generate PFFs or by isolating aggregates from animal models or patient brains. Injected PFFs or brain extracts are then causing accumulation in neurons projecting to the injection site. This model is unique in that it relies on triggering normal endogenous asyn to phosphorylate and accumulate rather than driving aggregation via elevating endogenous asyn. Asyn PFFs are commonly injected into the striatum. Several weeks following injections, asyn pathology can be visualized in prefrontal cortex, striatum, cortex and SN by immunolabeling for phosphorylated asyn (Figure 2K). Control injections of PBS or serum albumin do not result in those PD typical intraneuronal accumulation (Figure 2J).
The stereotaxic injection method can be challenging and expensive, and common errors can usually only be detected after the animals have been sacrificed. The capillary thickness of the blunt end can vary from 10-100 µm depending on the pulling method. If the capillary is too thin, it can clog while entering the brain and lead to a failure in injection (Figure 2L). In this particular case, the needle tract can be identified by scar tissue and uncleared blood cells while no effect of the injection is visible. Another common error is mistargeting (Figure 2M). With age, bregma can be difficult to determine, incorrect coordinates or reading errors can all lead to incorrectly placed injections. While targeting is less complicated in bigger animals (e.g., rats) and larger brain areas (e.g., striatum), they become increasingly more challenging in mice SN injections for example. Another common mistake is dosage. Control substances should not cause more than 10-15% cell death on their own. If expression of GFP (Figure 2N) or PBS injection is toxic, parameters need to be changed to ensure that pathology and cell death are specific to the substance injected.
Figure 1. Setup for stereotaxic surgery. (A) Schematic of a stereotaxic setup during survival surgery including digital reader and glass syringe which is further detailed on the right. (B) Skull anatomy of a rodent showing bone plates (frontal, parietal and occipital bones) with corresponding sutures resulting in bregma and lambda. (C,D) Adapted images from the Allen brain atlas webpage showing coronal sections of mouse striatum (C) and midbrain (D) with corresponding coordinate grid in mm. Images adapted from biorender. Please click here to view a larger version of this figure.
Figure 2. Histological examples of Parkinson's disease animal models. (A) Schematic of the orientation of a mouse brain. Blue line indicates location of coronal striatal section for B and C. Green line indicates location of coronal midbrain section in D and E. (B,D) Illustrations of coronal mouse brain section showing the striatal area in B and the midbrain area in D. Black box represents magnified ventral midbrain (substantia nigra) region in F-N. (C,E) Coordinates were tested for their accuracy prior to experiment by injecting 0.2 µL tryptophan blue. Brain was immediately taken out after surgery, frozen and cut on a microtome to analyze targeting. Examples for correct targeting of striatum and substantia nigra are shown in C and E, respectively. (F,G) Representative images of the 6-hydroxydopamine (6-OHDA) model are shown by immunolabeling for the dopaminergic marker tyrosine hydroxylase (TH) on nigral sections. 6-OHDA (4 µL of 3 µg/µL; G) or PBS as control (F) was injected into the medial forebrain bundle (MFB) and rats were sacrificed 6 weeks after surgery. Half an hour prior to injection, 25 mg/kg desipramine was given i.p. to prevent noradrenergic cell death. (H,I) Example of viral vector based animal model shows transduced cells in green (Immunolabeled against GFP transgene [control vector], H) or red (Immunolabeled for asyn, I), and DA neurons in white. AAVs with a serotype 6 (1 µL of 7 x 1013 viral genomes /mL each) were injected directly into the substantia nigra pars compacta (SN) and mice were sacrificed 4 weeks after surgery. (J,K) Representative images of rats injected with 8 µg of mouse asyn PFF (K) or with PBS as a control (J) were Immunolabeled for phosphorylated asyn (brown) and cell nuclei with cresyl violet (purple). Rats were injected into the striatum (2 x 2 µL deposits) and sacrificed 8 weeks after injection. Images adapted from Duffy et al.21. (L,M,N) Common errors of stereotaxic experiments are no viral solution injected (L, needle tract indicated by arrows), mistargeting (M, in this case too lateral) and control injection causes cell death (N, missing cells indicated by red circle). Transduced neurons are Immunolabeled in green for GFP and white for TH. Please click here to view a larger version of this figure.
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 difficult to recover from and additional wet food should be supplied.
Besides animal welfare, the main technical complication of stereotaxic injection is mistargeting. Thus, before starting each experiment, pilot animals should be used to verify the coordinates, even when they have been correct in previous experiments. Breeding, diet and age can influence head and brain shape and size and can lead to gross over/underestimation of coordinates. Since the targeting in smaller animals is more challenging, it is crucial to level the head properly to guarantee reproducible injection results. New technology where the stereotaxic instrument is connected to a computer program can help adjust coordinates for each animal by measuring specific points on top of the skull and reduce error. Errors in reading coordinates, especially after long hours of surgery, can also be reduced by digital tools and are worth the financial investment. In larger animals such as pigs and primates, MRI or CT scans are used to calculate more precise coordinates and reduce the chance of mistargeting.
Another challenging obstacle can be clogged capillaries during injection. Shape, size and thickness of each capillary can vary, especially if hand-pulled. If the glass is too thin, the capillary could break or bend during surgery, or clog by taking up tissue in the way of the injection path. If the capillary is too thick, it can disrupt and destroy many structures while going into the brain and leave scar tissue behind. Sutter Inc. therefore provides a cookbook (https://www.sutter.com/PDFs/pipette_cookbook.pdf) for making various shaped and sized capillaries although mostly optimized for electrophysiology. Newer pulling machines need to be operated manually for longer capillaries as used in surgeries. Another great alternative is to order pre-pulled capillaries with the precise specifications needed for your injections. Ideally the diameter of a blunt glass capillary can go up to 80-100 µm but shouldn’t be smaller than 20 µm while the length depends on your injection depth (DV coordinates).It is important to use glass as a material for your injection capillaries. Glass can be used with a finer gauge than metal needles and injectable substances are less prone to interact with or bind to glass.
As each animal is slightly different from one another, targeting will also vary from animal to animal creating a diverse cohort with a range of cell death and pathology. This diverse cohort can make it more difficult to interpret results or achieve a good N number. Consequently, each study has to be powered appropriately in order to be successful. More consistent strategies to develop in vivo models include genetic modifications to create transgenic animals. This method is more expensive and difficult in its beginning stages, even with CRISPR technology, but can pay off later on as these models have very little intra-animal variation and need minimal extra work. Furthermore, it is fairly uncomplicated to transfer transgenic animals from laboratory to laboratory making it a tool that can be easily available to many scientists. Weaknesses of the transgenic approach, at least in the field of Parkinson's disease, are the lack of dopaminergic cell death11, appropriate control lines in general and flexibility to change and adjust the model based on your hypothesis or novel evidence. Additionally, cross breedings will produce many more animals than needed for a set experiment. Once set up, stereotaxic animal models can be more cost efficient and flexible as toxicants, viral vectors and fibril strains can be altered more readily. Both methods have a valid place in the field of neurodegeneration and should be chosen by their appropriateness to address the scientific question asked.
The use of toxicants in PD animal models can result in consistent, severe and reproducible dopaminergic degeneration. Errors while using toxicants occur mainly while handling these substances. Most toxicants, such as 6-OHDA, are light and temperature sensitive and are prone to oxidation. This will render the substance ineffective even before the injection and can result in low to no toxicity. Therefore, solutions have to be freshly prepared adding ascorbic acid to prevent oxidation and have to be shielded from light exposure before and during the injection22. MPTP can be given systemically to animals causing substantial DA cell loss in the SN when given in acute doses over several days23. This model will also result in behavioral deficits but mostly lacks pathological aspects. Disadvantage of using MPTP are its lack of toxicity in rats and some mouse lines, and its neurotoxic risk among researchers and animal caretakers. Because of this risk, it is absolutely necessary to establish a proper working procedure before using MPTP in laboratories. While toxicants are a more straightforward tool, viral vectors need to be cloned and produced correctly in order to function efficiently. Cloning can be complicated as the plasmids contain two repetitive sequences necessary for packaging of the virus that could be lost recombinantly during replication. Additionally, packaging limitations only allow for an expression cassette to be 4.5 or 11 kbp for AAV or Lentivirus, respectively. This limitation can sometimes be bypassed by using multiple vectors or creative editing. Future vector engineering will undoubtedly be focused on extending the size of vector genomes. The production of viral particles can be difficult as the end product needs to be pure but also have a high titer24. Quality control is necessary before each batch of viral vector can be used to avoid increased inflammation or low expression. Furthermore, a dose-response curve should be generated as the GFP expressing vectors can also result in cell function abnormalities and cell death25. Similar to the vector models, quality control of PFFs is of utmost importance. The production of preformed fibrils has long been of great debate as different protocols result in different strains of fibrillar aggregates and may cause diverse pathological patterns26,27. Injections of poorly assembled fibrils will not result in the desired pathological phenotype. It is also worth mentioning that most laboratories use PBS as a control as injection of monomeric asyn can also cause several intracellular aggregates at longer time points28. To prevent initial pitfalls using these tools, viral vectors or PFF should be acquired from experienced researchers or companies. This can drastically reduce the initial failure rate and provide time to gain experience using stereotaxic injections.
Since stereotaxic injections is such a versatile method, new technology is constantly being developed to make this technique easier and more streamlined; from motorized robotic injection arms, over automated drilling to 3D reconstruction of the animal's head. This will allow for faster and more accurate injections in the future. Furthermore, new viral vectors are constantly being developed to improve spread and specificity while increasing their genome capacity. Additionally, new insights into PD but also neurodegeneration in general will help us improve our injectable tools to create these models. Recent scientific findings have let us to develop the PFF model and future discoveries, genetic or mechanistic, will help us advance PD models and guide us on our quest on finding a cure.
The authors have nothing to disclose.
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.
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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