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EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;
1. Preparation of reagents and supplies
<ol>
	<li>Determine L-3,4-dihydroxyphenylalanine methyl ester hydrochloride (levodopa or L-DOPA) and benserazide hydrochloride, a peripheral decarboxylase inhibitor (see Table of Materials) dose, rating frequency, and experimental timeline that is appropriate for the investigational question (Figure 1). 
	NOTE: Investigational questions can be posed to seek any number of answers, ranging from asking whether a specific therapy might reduce existing L-DOPA-induced dyskinesias (LID) to preventing LID induction. 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>
	<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. 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: The target dose is 12 mg/kg or 12 mg L-DOPA/1000 g body weight. Supplementary Information provides an 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.</li>
</ol>
2. Room and cage set up
<ol>
	<li>On the first day of L-DOPA treatment 3-4 weeks following 6-hydroxydopamine (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>
3. Levodopa injections and dyskinesia rating
<ol>
	<li>Subcutaneous injections of L-DOPA
	<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 1.3). 
		&#8203;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 environment.</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. Then, 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 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 time points, which should include the initial onset of LID behavior, peak behavior, and the phase of decline.</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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 Minutes Digital Timer</td>
			<td>Staples</td>
			<td>1111764</td>
			<td></td>
		</tr>
		<tr>
			<td>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 &#176;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 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&#39; 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/bens</td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 1" src="/files/ftp_upload/23103/23103_Figure1.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...

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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;
1. ...

Take a syringe filled with levodopa, a dopamine precursor, and a dopamine conversion inhibitor.
Inject this solution into the subcutaneous tissue of a restrained rat with Parkinson&#39;s disease, exhibiting damaged dopamine-producing neurons.
In the rat, the inhibitor binds to dopamine-producing enzymes and prevents the premature conversion of levodopa into dopamine.
Levodopa enters the bloodstream, crosses the blood-brain barrier via amino acid channels, and enters the nondamaged dopamine-producing neurons.
The enzymes in these neurons convert levodopa into dopamine, an excitatory neurotransmitter, and release it into the synapse.
Dopamine binds to its receptors on the post-synaptic neurons, triggering signal transmission.
Repeat the injections periodically to increase dopamine production.
Over time, excessive dopamine leads to overstimulation of its receptors and excessive neuronal signaling, which eventually disrupts the rat&#39;s body movements.&#160;&#160;
Place the rat in a cage and observe its behavior. The rat exhibits involuntary, repetitive body movements, known as dyskinesias, induced by levodopa.

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

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Neurodegenerative Diseases

53 Views. מקור: Caulfield, M. E., et al. אינדוקציה והערכה של דיסקינזיות המושרות על ידי לבודופה במודל חולדה של מחלת פרקינסון. J. Vis. Exp. (2021). סרטון זה מדגים את התהליך של גרימת דיסקינזיות המושרות על ידי לבודופה במודל חולדות פרקינסון על ידי מתן לבודופה לצד מעכב המרת דופמין. זריקות חוזרות ונשנות מעוררו...

Video: השראת דיסקינזיה הנגרמת על ידי לבודופה במודל חולדה של מחלת פרקינסון - Concept

Handwriting Analysis Indicates Spontaneous Dyskinesias in Neuroleptic Na&#239;ve Adolescents at High Risk for Psychosis

Growing evidence suggests that movement abnormalities are a core feature of psychosis. One marker of movement abnormality, dyskinesia, is a result of impaired neuromodulation of dopamine in fronto-striatal pathways. The traditional methods for identifying movement abnormalities include observer-based reports and force stability gauges. The drawbacks of these methods are long training times for raters, experimenter bias, large site differences in instrumental apparatus, and suboptimal reliability. Taking these drawbacks into account has guided the development of better&#160;standardized and more efficient procedures to examine movement abnormalities through handwriting analysis software and tablet. Individuals at&#160;risk for psychosis showed significantly more dysfluent pen movements (a proximal measure for dyskinesia) in a handwriting task. Handwriting kinematics offers a great advance over previous methods of assessing dyskinesia, which could clearly be beneficial for understanding the etiology of psychosis.

Handwriting analysis software significantly improves upon existing instruments measuring movement disorders. Individuals at&#160;risk for psychosis and healthy controls completed handwriting tasks to test for dyskinesia. Results suggest that youth at&#160;risk for psychosis exhibit dyskinesia and that handwriting analysis could significantly contribute to wider dissemination of early identification efforts

כתב יד ניתוח מציין dyskinesias ספונטני במתבגרים הנאיביים נוירולפטיות בסיכון גבוה לפסיכוזה

Growing evidence suggests that movement abnormalities are a core feature of psychosis. One marker of movement abnormality, dyskinesia, is a result of ...

JoVE Journal

Behavior

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

שליטה מחלת פרקינסון עם מסתגל עמוק גירוי המוח

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Medicine

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.

דירוג L-DOPA-המושרה על ידי דיסקינזיות במודל העכברוש החד-צדדי 6-OHDA-נגעים של מחלת פרקינסון

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

Transcript

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