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Sleep Medicine 8 (2007) 344–348

Animal models of restless legs syndrome

W.G. Ondo *, H.R. Zhao, W.D. Le

Department of Neurology, Baylor College of Medicine, Houston, TX 77030, USA

Received 13 March 2007; accepted 13 March 2007Available online 30 April 2007

Abstract

Restless legs syndrome (RLS) is a common disease with prevalence up to 10% in the general population. It is mostly a subjectivecondition, making animal models intrinsically difficult. General increased activity (urge to move) and limb movements consistentwith periodic limb movements of sleep, seen in most patients with RLS, are currently our best behavioral markers. Our best under-standing of human RLS demonstrates reduced central nervous system (CNS) iron stores and dysfunction of dopaminergic systems,which most likely involves the spinal cord. Based upon this knowledge, animal manipulations, including destruction of the A11 dien-cephalic–spinal tract and iron deprivation, have resulted in animal behavior consistent with RLS. Dopamine receptor type 3 knock-out mice also show general increased activity. Pharmacologic blockade of dopamine receptors in rodents has also caused movementsresembling periodic limb movements of sleep in older rodents but not in younger animals. More sophisticated animal modeling isneeded to facilitate our understanding of RLS.� 2007 Published by Elsevier B.V.

Keyword: Restless legs syndrome

1. Introduction

Restless legs syndrome (RLS) is a common diseasewith a prevalence up to 10% in the general population[1,2]. Onset can occur at any age, with a family historyof the disorder predicting an earlier age of onset.Women are somewhat more commonly affected thanmen. This syndrome is characterized by an intense urgeto move the limbs, especially the legs. Patients may notdescribe an actual sensation, but only a pure urge tomove, or focal akathisia [3]. This urge has a circadianpattern, which is most pronounced in the evening orduring the night and often relieved by walking. At least80% of RLS patients also suffer from the associatedperiodic limb movements of sleep (PLMS) and sleepdisturbance [4]. Another supportive feature is that the

1389-9457/$ - see front matter � 2007 Published by Elsevier B.V.

doi:10.1016/j.sleep.2007.03.010

* Corresponding author. Tel.: +1 713 798 7438; fax: +1 713 7986808.

E-mail address: wondo@bcm.tmc.edu (W.G. Ondo).

symptoms usually improve with low doses of dopamineagonists [5,6].

The underlying pathophysiology of RLS may involvedopamine transmission insufficiency. In patients withRLS, dopaminergic functional brain imaging studiesshow modest abnormalities, and the symptoms can bedramatically improved with the dopamine agonists andcan worsen with dopamine antagonists [7–9]. Further-more, there is a growing body of literature that suggestsa role for iron in RLS. Iron deficiency has been consid-ered a significant contributing cause of RLS. A stronginverse correlation between cerebrospinal fluid (CSF),serum ferritin levels, and RLS symptoms suggest thatiron deficiency is involved [10]. Imaging studies andneuropathological examination also suggests impairedbrain iron homeostasis in RLS [11–13]. Anatomically,the spinal cord is implicated in the primary involvementof the legs. Finally, the strongly nocturnal pattern ofsymptoms suggests some connection to circadian con-trol centers.

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To date, the pathophysiology of the syndrome hasnot been fully elucidated. Although current treatmentswith dopamine agonists symptomatically improveRLS, there is no cure for this disorder. In order to learnmore about RLS, a few animal models have been devel-oped based on current theories of pathogenesis.

1.1. 6-Hydroxydopamine (6-OHDA) lesioned rat model

Pathologic data for people with RLS is sparse. How-ever, the clinical features of RLS provide evidence topostulate pathoanatomic pathways. Foremost is thatRLS is extremely responsive to low doses of dopaminer-gic agents. This suggests that a dopaminergic system isimplicated in the pathogenesis of RLS. The preponder-ance of leg involvement suggests that spinal cord orpossibly the peripheral nerve involved. The circadianpattern reflects input from circadian control areas. Theunpleasant sensory component suggests dysfunction ofintrinsic antinociceptive mechanisms. It is believed thatpart of the diencephalospinal dopaminergic neuronsforms a sympatho-excitatory system. Perturbation ofthis system may result in clinical PLMS.

When these features are compiled, we postulated that aspecific diencephalospinal dopaminergic tract, originat-ing from the A11–A14 might explain some RLS features[14,15]. To test this hypothesis, Ondo et al. [16] lesionedrats in this area to determine whether any subsequentbehavioral responses were consistent with RLS. 6-OHDAwas stereotaxically injected into bilateral diencephalicA11 dopaminergic nuclei of Sprague–Dawley rats.Control animals underwent sham surgeries. The behaviorof these rats was observed several months later. Ratbrains were sectioned and examined whether the substan-tia nigra (SN) and/or ventral tegmental area (VTA) werealso affected by 6-OHDA injection in the A11 region. Toexamine the effect of the dopaminergic treatment on therats, the dopamine agonist pramipexole was used.

An overall reduction of about 50% of tyrosinehydroxylase (TH)-positive cells in the A11 region wasfound in rats after 6-OHDA lesions. Interestingly, thesecells proved more resistant to 6-OHDA lesioning thanother dopaminergic areas. The dopaminergic cells inthe SN and VTA were relatively preserved. Comparedto controls, the 6-OHDA-lesioned rats demonstrated alonger latency to sleep, less sleep time, and moreepisodes of standing upright during the observationperiods. Subsequently, pramipexole-treated rats showedless standing episodes and total standing time whencompared with untreated lesioned rats. There were nomovements that overtly resembled the PLMS seen inpeople.

Overall, the rats showed sleep disturbance and motorrestlessness, and these obvious behavioral changesimproved with dopamine agonist. All of these observa-tions were consistent with clinical RLS. Because this

study, however, only focused on the movement disorder,it could not assess the subjective aspects of RLS, such as‘‘unpleasant sensations’’ or ‘‘an urge to move.’’ This pre-liminary study was limited by its lack of electrophysio-logical sleep measures, intermittent observations, andsmall sample size.

In Qu et al.’s anatomical study [17], the fluorescenttracer Fluoro-Gold (FG) was used to investigate thepathway of diencephalic dopaminergic neurons innervat-ing the spinal cord in mice. The fluorescent tracer FGwas stereotaxically injected into the lumbar spinal cordof C57BL mice. The diencephalic sections then werestained with TH antibodies, and the FG tracer presentin the diencephalic dopaminergic neurons was examinedunder a fluorescence microscope. The results suggestedthat the diencephalic A11 dopaminergic neuronspossessed long axons extending over several segments,possibly traversing the entire length of the spinal cord.It was demonstrated for the first time that both A10and A11 group dopaminergic neurons project into thespinal cord in mice. Though increasing evidence hasdemonstrated dopaminergic diencephalic spinal neuronsare strongly implicated in RLS, lesioning this area mayonly partially mimic the clinical features of RLS.

1.2. 6-OHDA lesioned and iron-deficient mice

There is robust evidence to support that the underly-ing pathophysiology of RLS involves central nervoussystem (CNS) iron homeostatic dysregulation. CSF fer-ritin is lower in RLS cases [10], and imaging studies[12,13] show reduced iron stores in the striatum andred nucleus. Most importantly, pathologic data inRLS autopsied brains shows reduced ferritin staining,iron staining, and increased transferring stains, but alsoreduced transferrin receptors [18]. No pathology asidefrom iron abnormalities has been identified [19]. Subse-quent studies have demonstrated a strong correlationbetween serum ferritin levels and RLS symptom sever-ity: decreasing ferritin was associated with increasingRLS severity.

The relationship between iron and dopaminergic sys-tems is complex. Iron is a cofactor for TH that is therate-limiting enzyme for dopamine mechanism. Studiesin iron-deficient animals have also demonstrated adecrease in dopamine D2 receptor density [20]. Changesin basal ganglia dopamine metabolism associated withiron deficiency in rats are dependent on the time ofday. Using microdialysis techniques, an increase in DAand its metabolites were specifically associated withthe beginning of the dark period [21].

Therefore, both iron deficiency and dopaminergicsystems seem to play an important role in RLS. Todevelop an ideal animal model of RLS, Qu et al. [22]performed stereotaxic bilateral 6-OHDA lesions intothe A11 nucleus of C57BL/6 mice and fed them

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iron-deficient diets to observe whether these two mani-pulations could induce animal behavioral changes thatmimic clinical RLS.

Half of the mice received a regular diet and the otherhalf was fed a low-iron diet. One month after dietarymanipulation, 6-OHDA was stereotaxically injected intobilateral A11 nucleus in half of the dietary segregatedgroup. Iron levels in the serum were measured andbehaviors were observed at baseline, after dietary inter-vention and after 6-OHDA lesioning. Several pharma-cological interventions were performed subsequently.

Locomotor activities were carried out at differenttimes. One month after surgery, the animals were alsochallenged with three drugs known to improve or exac-erbate human RLS. These drugs included dopaminereceptors D2/D3 agonist ropinirole, D1 agonist SKF-38393, and D2 antagonist haloperidol. The locomotoractivities of animals were recorded after the administra-tion of the test drugs.

A serum iron assessment was obtained by tail vein indifferent periods. Brain and spinal cord samples werelater autopsied to measure the tissue iron concentration.After behavioral observation in all groups of animals,the sections from diencephalic regions and mesencepha-lon were stained for TH immunohistochemistry.

Pathologic examination demonstrated a markedreduction in A11 TH staining cells in mice injected with6-OHDA. Iron levels in serum, brain, and spinal cordwere significantly decreased after iron deprivation.Interestingly, CNS stores of iron were more reduced inthe animals that had A11 lesioning than those withoutlesioning, despite similar serum iron levels.

Locomotor activity was significantly increased inboth iron-deprived mice and A11-lesioned mice com-pared to controls. The combination of iron deprivationand A11 lesioning further augmented motor activity. Inaddition, the mice in the lesioned group were moreaggressive. Compared to controls, the increased activityin A11-lesioned mice with or without iron deprivationreturned to baseline after treatment with the D2/D3agonist ropinirole, which is used to treat RLS, but wasactually aggravated by the D1 agonist SKF38393. TheD2 antagonist haloperidol also worsened the locomotoractivity in general, but not specifically in the lesioned oriron-deprived animals.

This study demonstrated that iron deprivation alonecould increase the activity in mice. This behavior wassignificantly enhanced with 6-OHDA lesioned in A11dopaminergic neurons. Overall, this study supports thatthe manipulation of iron and dopaminergic systems canresult in behavioral changes in animals. Furthermore, itshowed that combining iron deficiency with A11 lesion-ing in mice can generally mimic the increased motoractivity seen in clinical RLS, which can be used as ananimal model of RLS and also in therapeutic trials.However, we have yet to test for the characteristic

human symptom of PLMS commonly seen in RLS,and we have yet to assess sleep in this model.

We recently corroborated these behavioral results in asecond study devised to determine the effects of irondeprivation and 6-OHDA A11 lesioning on spinal corddopamine receptors [23]. We found that the levels of D1mRNA and protein in the lumbar spinal cord wereincreased by ID, whereas D2/D3 mRNA and proteinswere not changed by dietary ID. A11 6-OHDA lesionsreduced D2/D3 mRNA and proteins in the lumbar spinebut had little effect on D1 mRNA. However, 6-OHDAlesions reduced the levels of D1 protein. ID and 6-OHDAlesions produced antagonistic effect on D1 protein levels.This dissociation between D1 mRNA and protein sug-gests a suppression of D1 mRNA transcription regulationafter A11 6-OHDA lesions. Statistical analysis found that6-OHDA lesions had marked effect on the decrease of thespecific D2 receptor binding of [3H]Spiperone. Further-more, iron deprivation and 6-OHDA lesions produced asynergistic effect on the decrease of D2 binding [3H]Spip-erone in the lumbar spinal cord of the mice. Specific D3receptor binding [3H]PD128907 was only down-regulatedby 6-OHDA lesions, which contrasts with previous workin the striatum, showing that ID reduced D2 family recep-tor density [24].

Data concerning the effects of pure iron deprivationin rodents is mixed. In contrast to our behavioralresults, Glover et al. [25] studied the activity pattern ofiron-deficient rats. They found that iron-deficient ratsappeared to have a decrease in total activity and a disor-der of diurnal rhythm. These were reversed by theadministration of iron. The discrepancy of these twostudies may be result from the different time of iron-defi-ciency diet and different animals. More consistent withour results, Dean et al. recently reported that iron-deprived mice showed increased wakefulness duringthe phase of their circadian period that would correlatewith RLS in humans [26]. They concluded that thisbehavior is consistent with RLS.

1.3. Dopamine D3 receptor knockout mice

Mammalian nervous system activity is under strongcircadian control from the hypothalamic suprachiasmat-ic nuclei (SCN) [27–31]. One SCN projection is to thedorsomedial hypothalamic (A11) dopaminergic nucleus.A11 neurons are the major source of spinal dopamine,with strong projections to the sympathetic preganglionicneurons in the intermediolateral nucleus [14,32]. There isextensive evidence for the existence of D1, D2, and D3receptors in the spinal cord [31]; however, the contribu-tion of these receptors to spinal reflex excitability is notwell known. Some previous data have demonstrated thepresence of D3 dopamine receptors in the rat spinal cordexpressed in highest densities in the superficial layers ofthe dorsal horn of the cervical and lumbar regions.

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The presence of D3 receptors in the superficial layers ofthe dorsal horn suggested that these receptors may playa role in sensory or nociceptive processing. A D3KOmouse displays hyperactivity, increased locomotoractivity, and hypertension. This phenotype looselyresembles features of patients with RLS that expressPLMS [32,33].

1.4. Animal model of PLMS

PLMS are periodic leg, more than arm, movementsthat predominately occur in stage 1 and stage 2 of sleep.Their frequency in humans ranges from 30 to 90 s. Sim-ilar movement called periodic limb movements whileawaking (PLMA) can occur during drowsiness. PLMScan occur as an isolated phenomenon but are often asso-ciated with RLS and represent the only purely objectivesign of this disorder [34].

It is known that the occurrence of PLMS and RLS inhumans increases with age and that a variety of dopami-nergic drugs reduce them. It is not clearly establishedwhether dopamine antagonists induce PLMS, but theyat least aggravate existing PLMS and clinically worsenRLS. Although these observations suggest that thedopaminergic system is involved in the genesis of PLMS,the pathophysiological mechanisms and biochemicalinteractions are not yet known. To elucidate the under-lying causes, an animal model of PLMS would be ofgreat value.

In order to delineate whether rats display periodichindlimb movements (PHLM) while they are asleep,Baier et al. [35] assessed hindlimb movements andsleep–wake behavior in this species. They investigatedthe occurrence of PHLM in a group of animals anddetermined whether the dopamine antagonist haloperi-dol would affect PHLM.

The experiments were performed on Wistar rats.Stainless steel screws were implanted epidurally in theskull to derive the electroencephalogram (EEG). Forelectromyographic (EMG) recording, stainless steelwires were inserted into the neck muscles bilaterallyand attached to the skull with screws. For movementdetection, two magnets were implanted subcutaneouslyin both hindlimbs. The animals recovered from surgerybefore data acquisition.

EEG, EMG, and hindlimb movements were recordedfrom light onset to dark onset. Haloperidol was injectedintraperitoneally at light onset. EEG and EMG signalswere amplified, filtered, and digitized as the previousreport [36]. A video recorder documented the behaviorof the animals for the entire time of data acquisition.Three vigilance stages – wakefulness, non-rapid eyemovement (NREM) sleep, and rapid eye movement(REM) sleep – were determined by the visual assessmentof EEG and EMG recordings as described elsewhere[37,38].

Hindlimb movements occurring during NREM sleepwere subdivided into those associated with an arousal(HLM) and those not associated with an arousal(HLM+). They defined PHLM in rats according tothe modified human criteria [39] as at least four consec-utive hindlimb movements with an inter-movementinterval of 5–60 s.

All animals exhibited comparable amounts of wake-fulness and NREM sleep, while young animals had sig-nificantly more REM sleep than the old ones.Haloperidol administration to the old animals did notaffect the overall time spent in any of the vigilancestages. All animals displayed some HLM and HLM+in NREM sleep, but most did not meet criteria forPHLM. The young animals showed especially fewPHLM. Haloperidol did not significantly influence anyof these parameters. Interestingly, the percentage ofold rats spontaneously displaying PHLM resemblesthe increased prevalence of PLMS in the elder patients.That study demonstrated for the first time that PHLMin sleep can occur spontaneously in rats. A clear effectof age on this phenomenon was seen, with only oldanimals displaying PHLM. To validate whether theobserved PHLM constitute a good model for humanPLMS or even RLS, their pharmacological propertiesneed to be characterized in a large number of PHLM-positive animals. CNS iron metabolism or deprivationwas not evaluated in this study.

2. Conclusion

In summary, several potential animal models for RLShave been developed, although not always for thatintended purpose. The key features involve iron depriva-tion and dopaminergic manipulation by genetic altera-tions or pharmacological intervention. The change ofsymptoms caused by dopamine agonists and antagonisthas been proved to be consistent with clinical RLS insome. However, it is difficult to completely mimic allkey factors related to RLS in any animal model, as thesensory component, or urge to move, can not be accu-rately assessed. It will be a major challenge to developa model in animals that can demonstrate this featureof the disorder.

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