Name: induction and assessment of levodopa-induced dyskinesias in a rat model of parkinson's disease
Uploaded: 2021-10-14T14:30:00.000+00:00
Duration: 5 min 51 s
Description: Watch this Scientific Journal Video about Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease at JoVE.com

We want to acknowledge the struggles of all those with Parkinson&#39;s disease and the strength and resilience they show every day, especially the beloved father of KSC, Mark Steece. The work represented here was supported by the National Institute of Neurological Disorders and Stroke (NS090107, NS110398) and the Parkinson Disease Foundation International Research Grant Program, now the Parkinson Foundation.&#160;We also would like to acknowledge Molly VanderWerp for her excellent editorial assistance.

The animals presented here were maintained and handled in compliance with the institutional guidelines. All animal procedures were approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC) in compliance with federal and state regulations.
1. Drug-free confirmation of 6-OHDA lesion status
<ol>
	<li>Postural Tail hang test11,12,13 
	NOTE: Assess lesion status at least 1 week following 6-OHDA&#160;lesion induction (See9,10,35&#160;for details on lesioning) in experimental subjects (e.g., male or female, adult Sprague Dawley or Fisher 344 rats).&#160;
	<ol>
		<li>Suspend the rat approximately 6 cm above its cage, firmly holding at the base of the tail, for ~5 s.</li>
		<li>Record the direction of body contortion as + for a successfully lesioned animal twisting contralateral to the lesioned side and - for lack of twisting or twisting in both directions.</li>
		<li>NOTE: These tests are optional but recommended. See14,15,16,17 for additional drug-free testing options/variations.</li>
	</ol>
	</li>
	<li>Step adjusting drag test (adapted from16) 
	NOTE: Assess lesion status at least 1 week following unilateral 6-OHDA lesion induction in experimental subjects (e.g., male or female, adult Sprague Dawley or Fisher 344 rats).
	<ol>
		<li>Hold the rat by the base of its tail, elevating the back feet off the surface by ~6 cm; drag backward across a flat, smooth but not slippery surface, ~75 cm, over 5&#8211;10 s.</li>
		<li>Observe and record the number of tapping/step adjusting movements of each forepaw over three repeated tests.</li>
		<li>Score the subject as + for successful unilateral lesioning when 0&#8211;2 forepaw taps are observed contralateral to the lesioned side, together with rapid tapping (~10 taps) from the forepaw ipsilateral to the lesioned side (e.g., animals unilaterally lesioned on the left side show tapping deficit (0&#8211;2 taps) with the contralateral right forepaw).</li>
		<li>Conversely, score moderate to rapid tapping (5&#8211;10 taps) from both forepaws as - to indicate incomplete or unknown lesion status. 
		NOTE: An anxious animal can show rapid tapping/step adjusting even if successfully lesioned. If this is suspected, place the rat back in their home cage and re-test &#8805;30 mins later.</li>
	</ol>
	</li>
</ol>
2. Preparation of reagents and supplies
<ol>
	<li>Determine L-3,4-dihydroxyphenylalanine methyl ester hydrochloride (levodopa or L-DOPA)&#160;and benserazide hydrochloride, a peripheral decarboxylase inhibitor (see Table of Materials) dose, rating frequency, and experimental timeline that is appropriate for the investigational question12,18,19,20 (Figure 1). 
	NOTE: Investigational questions can be posed to seek any number of questions ranging from asking whether a specific therapy might reduce existing LID or prevent induction of LID. They can also explore whether therapeutic efficacy is dependent on the dose of levodopa or whether LID expression and/or therapeutic efficacy varies depending on the sex, species, and age of the subject.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62970/62970fig1v3.jpg" /> 
Figure 1: Example of treatment timeline. Example L-DOPA dose-escalation timeline of 12 weeks in total length, with 8 weeks of L-DOPA injections beginning 3 weeks after 6-OHDA lesioning and 4 weeks following experimental treatment. In this example, L-DOPA is subcutaneously injected 5x per week (Monday&#8211;Friday) at approximately the same time each day, for 2 weeks at each prescribed L-DOPA dose (3, 6, 9, and 12 mg/kg). Behavioral LID ratings take place on days 1, 6, and 10 of each L-DOPA dosage level. <a href="https://www.jove.com/files/ftp_upload/62970/62970fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Weigh rats weekly to calculate the appropriate drug quantity based on the ongoing weight changes during the study. 
	NOTE: Due to increased activity in LID+ rats, there is potential for weight loss with long-term L-DOPA treatments.&#160;If weight loss occurs provide rats with nutritionally complete, highly palatable treats (see Table of Materials) following L-DOPA injections.</li>
	<li>Calculate the amount of L-DOPA and benserazide required for each weekly concentration, weighing out lyophilized aliquots for each day of injection, and storing in combination for 1&#8211;2 weeks at -20 &#176;C in glass amber vials until the day of treatment. 
	NOTE: Target dose is 12 mg/kg or 12 mg L-DOPA/1000 g body weight. Example of calculations for determining the amount of L-DOPA and saline needed for each day of a week using 12 mg L-DOPA/kg body weight at an injection volume of 1 cc/kg of rat weight is given in Supplementary File 1.</li>
</ol>
3. Room and cage set up
<ol>
	<li>On the first day of L-DOPA treatment 3-4 weeks following 6-OHDA lesion surgery, transfer rats to single housing, including IACUC-approved enrichment.</li>
	<li>Maintain in single housing throughout the study to avoid peer interference with behavioral assessments.</li>
	<li>On LID rating days, place the home cages on a steel wire rack, turned at an approximately 45&#176; angle for optimal viewing of the rat (Figure 2A). Flip the identification tags (Figure 2B) upward and remove water bottles, food racks, and all types of enrichment in the cage (Figure 2C) to avoid interference with behavioral assessments.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62970/62970fig02.jpg" /> 
Figure 2: Example of the cage set up for LID ratings of large-scale rat experiments. (A) Multiple cages can be set up for LID rating using large metal racks that allow for optimal viewing of each animal. Cages should be spread apart at a 45&#176; angle with ID cards flipped upward (B), food, water bottles, nesting materials, and other enrichment removed to limit visual obscureness of the rat and distractions to the rat while examining dyskinetic behaviors (C). The metal racks need to be a few feet away from any wall to allow the rater to examine the rat at the front or back of the cage as needed. It is essential to label enrichment apparatuses (e.g., C- red rat retreat houses) with individual animal IDs to replace them into the same cage from which they came. This is particularly important when using animals of different sexes not to increase stress to the experimental subjects. <a href="https://www.jove.com/files/ftp_upload/62970/62970fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Levodopa injections and dyskinesia rating
<ol>
	<li>Subcutaneous injections of L-DOPA21,22
	<ol>
		<li>Immediately preceding the daily injection of L-DOPA, add the appropriate volume of sterile saline to the pre-weighed lyophilized L-DOPA and benserazide mix in the amber vial and shake well for 10 s (step 2.3). 
		NOTE: Target injection volume is 1 mL/1000 g body weight (with 12 mg L-DOPA per mL). The volume of the sterile saline will depend on the number of animals per study.</li>
		<li>Fill individual syringes (e.g., 1.0 or 0.5 mL with 26 G needle) with the required volume for each animal (1 mL/kg rat weight) and label each syringe with individual animal identification. 
		NOTE: Keep the filled syringes protected from light in sterile pouches until the time of injection. L-DOPA rapidly oxidizes in the presence of oxygen and light in an aqueous environment23,24,25,26.</li>
		<li>Bring the first cage to the injection bench.</li>
		<li>Remove the rat from its cage and place it on the injection surface.</li>
		<li>Gently restrain the head and shoulders against the surface on which the rat is resting with the palm of the non-dominant hand.</li>
		<li>Gently, scruff the skin on the back overlying the scapulae with the thumb and forefinger of the non-dominant hand, inject L-DOPA volume with the dominant hand into the subcutaneous space between/below the fingers, keeping the needle as parallel to the body as possible to avoid intramuscular injection. 
		&#8203;NOTE: The rats are not anesthetized before injection.</li>
		<li>Dispose of each used individual syringe in a sharp&#39;s container.</li>
		<li>Replace the rat into its individual cage and add nutritionally complete treats except on LID rating days to avoid interference with behavioral assessments until after ratings are complete.</li>
		<li>Set the timer for 1&#8211;2 min depending on the rating time desired and the number of rats in the study on rating days. Retrieve the next cage and inject the next rat when the timer indicates.</li>
		<li>Repeat this, injecting one rat every 1&#8211;2 min, until all the rats are injected.</li>
	</ol>
	</li>
	<li>Levodopa-induced dyskinesia rating post-injection
	<ol>
		<li>Rate the intensity (Table 1) and frequency (Table 2) of dystonic and hyperkinetic dyskinesia movements at the desired number of timepoints, which should include the initial onset of LID behavior, peak behavior, and the phase of decline (see Supplementary File 2 for an example of LID rating log sheet).</li>
		<li>For male and female adult Sprague Dawley or Fisher 344 rats, and a sample size of N = 40 rats, begin the dyskinesia ratings 20 min after the first L-DOPA injection, and then at 50 min intervals until 220 or 270 min post-injection, depending on when LID behaviors have discontinued in 90%&#8211;100% of rats.</li>
		<li>If using 1 min rating intervals, set a timer for 1 min. Rate the first rat for one minute. Move to the next rat and rate it for 1 min. Continue through all the rats, rating for 1 min intervals.</li>
		<li>Have a timer positioned next to the cage visibly so that the LID behavior intensity (Table 1) can be observed while estimating the frequency of any given behavior (Table 2) during the rating period.</li>
		<li>After ratings for the first time point are completed start again with the first rat at the next time point (e.g; 70 min post-injection) and continue at the desired interval (e.g., every 50 min) until all time points are completed. 
		NOTE: Due to the overlap of L-DOPA injection and LID rating tasks, two persons are needed on the rating days, one for injecting and one for behavioral ratings.</li>
	</ol>
	</li>
</ol>

No conflicts of interest were declared.

Presented here are details for the reproducible induction and rating of LIDs in a parkinsonian rat model following unilateral 6-OHDA lesioning of the nigrostriatal DA system. While it was once thought that rodents did not develop LID and that rotational asymmetry may be the analog of LID in rats31, rat and mouse models have been characterized over the past two decades and are a well-accepted tool for LID research15,32,<sup class...

Although it has been more than 50 years since levodopa (L-DOPA) was first introduced as a treatment for individuals with PD1,2, it remarkably remains the most effective therapy for parkinsonian motor symptoms. The clinical motor symptoms associated with PD stem from the loss of dopamine (DA) neurons in the substantia nigra (SN) pars compacta, resulting in the dramatic decrease in available dopamine in the striatum. L-DOPA effectively restores striatal DA levels, resulting in motor benefit early in the disease3,4. Inopportunely, with long-term treatment, most individuals with PD will develop&#160;L-DOPA induced dyskinesias (LID), including chorea, dystonia, and athetosis, which often significantly impact activities of daily living5,6,7.
While several behavioral models of LID in rodents exist, differences in modeling and behavioral assessment of LIDs have called into question the reproducibility of results between labs as well as the reliability of these experimental tools for pre-clinical PD research. Developed in association with a clinical movement disorder specialist8, the current protocol is a straightforward method for LID induction and rating and is appropriate for use in a rat model of PD utilizing 6-hydroxydopamine (6-OHDA)-induced unilateral nigral lesioning9,10. The LID rating scale provided here includes scoring for both the intensity and frequency of dyskinetic behavior in various individual body parts. Pertinent information regarding workflow optimization of experiments and the appropriate care and handling of parkinsonian and dyskinetic animals is also provided.

<table><tbody><tr><td>&amp;nbsp;100 Minutes Digital Timer</td><td>Staples</td><td>1111764</td><td></td></tr><tr><td>&amp;nbsp;Compass CX Compact Scale</td><td>Ohaus</td><td>30428202</td><td></td></tr><tr><td>5-(2-aminoethyl)-1,2,4-benzenetriol, monohydrobromide</td><td>Cayman Chemicals</td><td>25330</td><td>6-OHDA is a catecholaminergic neurotoxin that is used to induce dopaminergic lesions and parkinsonian symptoms in rodents.</td></tr><tr><td>Allentown cages</td><td>Allentown, LLC</td><td>Rat900</td><td>Allentown cages provide the ability to view the rats from all sides.</td></tr><tr><td>BD Allergist Trays with Permanently Attached Needle</td><td>BD</td><td>BD 305540</td><td>For subcutaneous L-DOPA injections</td></tr><tr><td>Benserazide hydrochloride</td><td>Sigma-Aldrich</td><td>B7283</td><td>Benserazide is a peripheral decarboxylase inhibitor used with L-DOPA to to induce dyskinesia in rodent models of PD.</td></tr><tr><td>Glass amber scintillation vials</td><td>Thermo Scientific</td><td>B7921</td><td>Used for storage of L-DOPA/benserazide at -20 &amp;deg;C until mixed with sterile saline.</td></tr><tr><td>L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride</td><td>Sigma-Aldrich</td><td>D1507</td><td>L-3,4-Dihydroxyphenylalanine methyl ester is a precursor to&amp;nbsp;L-DOPA that crosses the blood-brain barrierand use to treat parkinsonian symptoms in rodents.</td></tr><tr><td>Paper Mate Sharpwriter Mechanical Pencils</td><td>Staples</td><td>107250</td><td></td></tr><tr><td>Rodent nutritionally complete enrichment treats</td><td>Bio-Serv</td><td>F05478</td><td></td></tr><tr><td>Round Ice Bucket with Lid, 2.5 L</td><td>Corning</td><td>432129</td><td></td></tr><tr><td>Standard Plastic Clipboard</td><td>Staples</td><td>1227770</td><td></td></tr><tr><td>Steel wired 6&#039; long movable shelving units</td><td>Uline</td><td>H9488</td><td>Width/Height can be adjusted to need/number of rats per experiment</td></tr><tr><td>Sterile Saline 0.9%</td><td>Covidien/Argyle</td><td>1020</td><td>For mixing with L-DOPA/benserazide prior to subcutaneous injections.</td></tr></tbody></table>

induction and assessment of levodopa-induced dyskinesias in a rat model of parkinson's disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

LIDs in parkinsonian rats can manifest as a range of abnormal involuntary movements (AIMs), including dystonic, hyperkinetic, and stereotypic behaviors. LID rating criteria for such behaviors are presented here to include both intensity (Table 1) and frequency (Table 2). This provides an overall LID severity score for each rat that reflects both the quality (intensity) and quantity of time spent engaging (frequency) in these behaviors at each rating timepoint. The final LID severity scor...

Watch this Scientific Journal Video about Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease at JoVE.com

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

induction-assessment-levodopa-induced-dyskinesias-rat-model-parkinson

Research

JoVE Journal

Neuroscience

3.6K Views.  Michigan State University. This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson's disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Video: Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Behavior

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Medicine

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and dementia. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can lead to some Parkinson&#8217;s-like symptoms by destroying dopaminergic neurons in the substantia nigra of the brain. It has been thus used to establish PD models in various animal studies. Here, mice receive MPTP injections (20 mg/kg/day) for seven days and the behavioral tests are performed on the eighth day. This model is adapted efficiently in the study of PD. The behavioral tests here include the cylinder test and the open field test. The cylinder experiment is used to detect the animals&#8217; ability to lift their front paws when put into a different environment. As the PD model mice show arching&#8212;the mouse arches its back&#8212;the number of paw liftings decrease. This test is easy to execute. The open field test is used to detect the amount of time the mice spend on running, walking, and remaining immobile. We analyze animals&#8217; movements in open field using software and obtain data. Lastly, we use L-DOPA, one of the most commonly used PD drugs, as one example to show how to apply this model to the study of PD drugs. Our results indicate that MPTP neurotoxicity induces motor deficit which can be mitigated by L-DOPA.

We describe establishment of a murine model for Parkinson's disease using MPTP, and behavioral assessments using cylinder and open field tests to measure motor function. We then use L-DOPA as one example to show how to apply this model in the study of PD drugs.

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Biology

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do not present a motor phenotype resembling the clinical aspects of the human disease. Pharmacological mouse models allow for a more direct study of the pathomechanisms and their effect on the behavioral phenotype. Osmotic pumps connected to brain cannulas open up the possibility of creating pharmacological mouse models via local and chronic drug delivery. For the hereditary movement disorder of rapid-onset dystonia-parkinsonism, the loss-of-function mutation in the &#945;3-subunit of the Na+/K+-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the &#945;3-subunit in the basal ganglia and the cerebellum, which are the two brain structures believed to be heavily involved in the pathogenesis of rapid-onset dystonia-parkinsonism, a bilateral cannula is stereotaxically implanted into the striatum and an additional single cannula is introduced into the cerebellum. The cannulas are connected via vinyl tubing to two osmotic pumps, which are subcutaneously implanted on the back of the animals and allow for the chronic and precise delivery of ouabain. The pharmacological mouse model for rapid-onset dystonia-parkinsonism carries the additional advantage of recapitulating the clinical and pathological features of asymptomatic and symptomatic mutation carriers. Just like mutation carriers of rapid-onset dystonia parkinsonism, the ouabain-perfused mice develop dystonia-like movements only after additional exposure to stress. We demonstrate a mild stress paradigm and introduce two modified scoring systems for the assessment of a motor phenotype.

We provide a protocol to generate a pharmacological DYT/PARK-ATP1A3 dystonia mouse model via implantation of cannulas into basal ganglia and cerebellum connected to osmotic pumps. We describe the induction of dystonia-like movements via application of a motor challenge and the characterization of the phenotype via behavioral scoring systems.

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

The 6-hydroxydopamine Rat Model of Parkinson's Disease

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and dopaminergic striatal deficit. Animal models have been widely used to simulate human pathology in the laboratory. Rodents are the most used animal models for PD due to their ease of handling and maintenance. Moreover, the anatomy and molecular, cellular, and pharmacological mechanisms of PD are similar in rodents and humans. The infusion of the neurotoxin, 6-hydroxydopamine (6-OHDA), into a medial forebrain bundle (MFB) of rats reproduces the severe destruction of dopaminergic neurons and simulates PD symptoms. This protocol demonstrates how to perform the unilateral microinjection of 6-OHDA in the MFB in a rat model of PD and shows the motor deficits induced by 6-OHDA and predicted dopaminergic lesions through the stepping test. The 6-OHDA causes significant impairment in the number of steps performed with the contralateral forelimb.

The 6-hydroxydopamine (6-OHDA) model has been used for decades to advance the understanding of Parkinson's Disease. In this protocol, we demonstrate how to perform unilateral nigrostriatal lesions in the rat by injecting 6-OHDA in the medial forebrain bundle, assess motor deficits, and predict lesions using the stepping test.

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in ...

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). The most common pattern observed in the clinic is the peak-dose dyskinesia which consists of clinical manifestations of choreiform, dystonic, and ballistic movements. The 6-hydroxydopamine (6-OHDA) rat model of PD mimics several characteristics of LIDs. After repeated L-DOPA administration, 6-OHDA-lesioned rats exhibit dyskinetic-like movements (e.g., abnormal involuntary movements, AIMs). This protocol demonstrates how to induce and analyze AIMs in 6-OHDA-lesioned rats with 90%-95% dopaminergic depletion in the nigrostriatal pathway. Repeated administration (3 weeks) of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) can induce the development of AIMs. The time course analysis reveals a significant increase in AIMs at 30-90 min (peak-dose dyskinesia). Rodent models of LIDs are an important preclinical tool to identify effective antidyskinetic interventions.

Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease.

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). ...

Induction of Levodopa-Induced Dyskinesias in a Rat Model of Parkinson's Disease

Source: Caulfield, M. E., et al. <a href="https://app.jove.com/v/62970">Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson&#39;s Disease.</a> J. Vis. Exp. (2021).
This video demonstrates the process of inducing levodopa-induced dyskinesias in a Parkinson&#39;s rat model by administering levodopa alongside a dopamine conversion inhibitor. Repeated injections overstimulate the brain neurons, resulting in motor dysfunction.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;
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