Animal studies in restless legs syndrome

Animal Studies in Restless Legs Syndrome

Paul Christian Baier, MD,1,2* William G. Ondo, MD,3 and Juliane Winkelmann, MD4,5,6

1Department of Psychiatry and Psychotherapy, Christian-Albrechts-University Kiel, Kiel, Germany2Department of Clinical Neurophysiology, Georg-August-University Gottingen, Gottingen, Germany

3Department of Neurology, Baylor College of Medicine, Houston, Texas4Max-Planck Institute of Psychiatry, Munich, Germany

5Institute of Human Genetics, GSF National Reseach Center for Environment and Health, Munich, Germany6Technical University, Institute of Human Genetics, Munich, Germany

Abstract: Although restless legs syndrome (RLS) is a commondisorder that has been studied thoroughly in the past decades,the underlying pathophysiology is still not fully understood.However, some attractive hypotheses on the pathogenesis ofthe disorder have been forwarded. Animal models are an im-portant tool to verify hypotheses and to dissect out the detailsof pathophysiological mechanisms. Ideally they might serve the

development of future treatment strategies. This review dis-cusses the general and specific prerequisites necessary for theestablishment of animal models for RLS and summarizes theapproaches that have been made. © 2007 Movement DisorderSociety

Key words: restless legs syndrome; RLS; periodic limbmovements; PLM; animal; model.

ANIMAL MODELING—GENERALCONSIDERATIONS

Although a “perfect” animal model would reproduceall key features of the human disorder being modeled,this is neither realistic nor necessary. There are usefulanimal models of human disorders that do not reproduceall features of the human condition, in fact, some do notreproduce any. The quality of an animal model is char-acterized by its reliability, and by its validity.1,2 There arethree concepts of validity that can be distinguished: facevalidity, predictive validity, and construct/etiological va-lidity. In short, face validity is high if the model exhibitsa behavioral or biological syndrome that meets typicalcriteria used to define the syndrome in humans. A mod-el’s face validity is proportional to the degree it meets allof the criteria. Sometimes animal models that onlypresent with a subset of a human syndrome prove usefulfor understanding at least that part. Predictive validitymeans that the model allows to make an extrapolation of

a particular experimental manipulation from the modelspecies to humans, e.g. if it allows to correctly predict theefficacy of a putative treatment. High predictive validitycan also be achieved when the behavioral phenotype ofthe model does not mimic the human disorder. Construct/etiological validity means that the model is derived froma cause known to cause the syndrome in humans and itsprocedures are theoretically sound. This requires a rea-sonably well developed knowledge of putative patho-physiology. The degree of this validity depends on thedegree of parallel between the putative mechanisms inthe animal and the pathology observed in humans.

ANIMAL MODELING—SPECIFICCONSIDERATIONS

To model restless legs syndrome (RLS) in animals,two steps are necessary: (1) The essential constituents ofa (behavioral) animal phenotype of RLS have to bedefined. (2) Knowledge and hypotheses on the etiologyof RLS have to be applied to animals, to see if anexpected animal phenotype can be observed.

DEFINING AN ANIMAL PHENOTYPE OF RLS

RLS is a subjective clinical diagnosis that is based onthe assessment of symptoms in patients. The definition of

*Correspondence to: Dr. Paul Christian Baier, Department of Psychi-atry and Psychotherapy, Christian-Albrechts-University Kiel, Ni-emannsweg 147, 24105 Kiel, Germany. E-mail: [email protected]

Received 7 January 2007; Revised 25 April 2007; Accepted 30 April2007

Published online 26 June 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mds.21605

Movement DisordersVol. 22, Suppl. 18, 2007, pp. S459–S465© 2007 Movement Disorder Society

S459

a behavioral animal RLS phenotype, however, must bebased on observable and measurable symptoms and fea-tures of human RLS. Although none of the minimaldiagnostic criteria3 can be measured directly, some be-havioral changes can be expected from the symptomspresent in and required for the diagnosis of RLS. Onemajor challenge is that all those observable (behavioral)features of RLS individually are very nonspecific andcan only achieve a high face validity, when their obser-vation is combined.

An urge to move the legs, usually accompanied orcaused by uncomfortable and unpleasant sensations inthe legs. Unpleasant sensory symptoms in the limbs ofexperimental animals could result in an increased atten-tion to the affected limb, demonstrated by licking orscratching. Locomotor activity assessments can serve asan indirect parameter for the “urge to move”. An increasein locomotor activity is probably very sensitive to anurge to move. However, as it may occur in many condi-tions, it has to be stressed, that it alone is a nonspecificsign for a behavioral RLS phenotype.

The urge to move or unpleasant sensations begins orworsen during periods of rest or inactivity such as lyingor sitting and the urge to move or unpleasant sensationsare partially or totally relieved by movement, such aswalking or stretching, at least as long as the activitycontinues.

In an experimental paradigm allowing animals tochoose between an environment with restricted mobilityand a slightly aversive environment with unrestrictedmobility, a preference for unrestricted mobility would beexpected in an animal RLS phenotype. Additionally,reduced amounts of wakeful inactivity, as determined bycombined sleep and activity assessments, would beexpected.

The urge to move or unpleasant sensations are worsein the evening or night than during the day or only occurin the evening or night. A circadian variation of theaforementioned parameters with an increase of impair-ment towards “the evening or at night” (i.e. the end ofactivity/beginning of the resting phase) could be ob-served in animals, if behavioral assessments were per-formed at several time points or continuously over com-plete diurnal cycles. Increases of locomotor activity onlyin certain phases of the diurnal cycle would hence bemuch more specific for a behavioral RLS phenotype thanthe observation of a generalized increase in locomotoractivity alone.

Supportive Criteria

The supportive diagnostic criteria of RLS (1) sleepdisturbance, with increased wake times and (2) periodic

limb movements (PLM) could be recorded polysomno-graphically, although it has to be considered that PLMmay be a feature unique to homo sapiens. There isevidence that PLM are modulated by another periodicsleep phenomenon called the cyclic alternating pattern(CAP) of sleep.4-6 Furthermore, CAP rates seem to beincreased in RLS patients (personal communication withR. Ferri). As there is an analogue to CAP that can beanalyzed in rats7,8 changes in the microstructural aspectsof sleep-wakefulness cycle could become a feature ana-lyzed in an animal model of RLS.

From the supportive diagnostic criteria one would alsoexpect a change of behavior after pharmacological inter-ventions known to relieve or exacerbate symptoms inhuman RLS.

Associated Features

Associated physiological features, extraneous to thecardinal symptoms included in the diagnostic criteria,could be utilized to further characterize a possible RLSphenotype.

Changes in spinal cord excitability9 and in pain10 andthermal sensitivity11,12 as seen in RLS patients, couldtheoretically be assessed in animals. However, it has tobe pointed out that the most commonly used tests forrodents (e.g. hot-plate and tail-flick-test) are likely to betoo insensitive to replicate the mild disturbances in deepsensory afferent transmission reported in RLS patients.

Although results are somewhat conflicting in details,several studies found evidence for a diffuse subclinicaldisinhibition of the motor cortex in RLS patients.13-16

This mild and nonspecifically increased cortical excit-ability, however, would be difficult to observe in animalexperiments.

On the basis of the epidemiological observation of anincreased association of RLS and hypertension,17,18 Cle-mens et al.19 suggested a common spinal cord mecha-nism. Circadian changes in blood pressure could beassessed in experimental animals and contribute to thecharacterization of an animal phenotype.

Overall, none of these physiological measures arespecific for RLS, and therefore are of limited utility whenobserved in isolation.

Biological Features

Animal models can be established on the basis ofgeneral types of biological abnormalities seen in thecondition to be modeled. These biological changes couldinclude abnormal blood test results and occur even whena behavioral phenotype is hard to define. There is strongevidence that reduced CNS iron content is associatedwith the RLS. Therefore changes in brain iron status after

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manipulations of peripheral iron metabolism could be apotential readout in an animal model of RLS (see later).

APPLYING HYPOTHESES

So far, none of the animal experimental approacheshas observed a complete “RLS-phenotype” in animals.However, on the basis of different pathophysiologicalhypotheses some animal experimental studies were per-formed and some of the aforementioned phenotypicalfeatures observed.

The Iron-Deficiency Hypothesis

Several human studies found a relationship betweenlow serum and CSF ferritin values and RLS symp-toms.20-23 The hypothesis of impaired iron metabolism inthe CNS in at least a subgroup of RLS patients is sup-ported by reports of reduced brain iron content in RLSpatients post mortem,24 with magnetic resonance imag-ing25,26 and by ultrasound findings indicating low iron inthe substantia nigra of RLS patients.27,28 More severeperipheral iron loss is associated with an increased riskof RLS. Nevertheless not all patients with disturbedperipheral iron metabolism suffer from RLS. Thereforesome additional factors, such as an interindividual vari-ation in the relation between peripheral and central ironcontent must contribute. The environmental iron chal-lenge relation to RLS may be both a dose and subjectvariable response.

Behavioral observations of rodents with altered ironstatus are somewhat inconclusive. Several authors reportthat severe iron deficiency leads to reduced motor activ-ity in rodents,29-31 a behavior that would contradict anRLS animal phenotype. It should be noted that severesystemic iron deficiency, such as induced in these stud-ies, is unlike that usually seen with RLS. A single reportthat iron deficiency reverses diurnal patterns of thermo-regulation and motor activity in rats29 could not be rep-licated in another.30 However, in the latter study severaldegrees of iron deficiency were investigated. It wasfound that rats with marginal iron nutriture were gener-ally more active in the light, i.e. their resting phase,compared to iron-adequate and severely iron-deficientanimals.30 In agreement with this, a recent study by Deanet al.32 reported an increase of wakefulness in the 4 hourspreceding the resting phase (i.e. the “evening”) of iron-deficient mice. Total wakefulness over 24 hours re-mained unchanged. Both studies would be consistentwith the circadian variations of symptoms in humanRLS, with an increase of complaint at evening and in thenight.

There are few studies focusing on potential biologicalmarkers of RLS in iron-deprived animals. Thy-1 is a cell

adhesion molecule that plays a regulatory role in thevesicular release of neurotransmitters. Transient iron-deprivation from gestational day 5 to weaning at postna-tal day 21 led to a reduction of Thy1 in the brainhomogenates of rats.33 The authors reported in the samestudy, that Thy 1 was reduced to less than half of controlsin the post mortem substantia nigra of 4 patients withprimary RLS. In another study, the effect of dietary irondeprivation on dopamine receptor expression was inves-tigated.34 In this study, severely iron-deficient and ane-mic rats showed reduced densities of D1 and D2 recep-tors in the caudate-putamen and decreased D2 receptordensities in the nucleus accumbens. However, dopaminereceptor binding imaging studies in humans with RLSare inconclusive, with reduced,35,36 normal,37,38 and in-creased striatal D2-receptor-binding.39 Whether the find-ing in those-severely—iron deprived —rats resemblesbiological changes in human RLS is therefore unclear.

It seems that the dietary iron deficient model, if severesystemic iron deficiency is avoided, to date is the onlyapproach that has data producing behavioral changes thatmeet the critical circadian change in activity and sleep-wake pattern. However, further investigations are neces-sary, in which motor activity is investigated in conjunc-tion with sleep changes. Also, the predictive validity ofthis putative animal model for RLS has to be tested withtreatments known to be effective in alleviating the symp-toms of RLS, including dopaminergic agonists.

The A11-Hypothesis

The specific anatomic involvement of RLS within thenervous system is not known, however, several clinicalfeatures allow speculation. First, RLS responds dramat-ically to dopaminergics. Second, the primary leganatomy, the frequent occurrence of RLS after spinalcord lesions40,41 and neurophysiological observations9

strongly implicate spinal cord involvement. Third, thesensory component suggests some form of afferent dis-inhibition.10 Fourth, associated PLM are influenced fromthe autonomic nervous system. Fifth, circadian fluctua-tion of symptoms with a worsening at night, suggestssome connection to circadian control centers.42 Thesefeatures have led to the hypothesis that the diencephalicspinal tract (A11 dopamine cell cluster) may be involvedin the pathogenesis of RLS. It is dopaminergic, subservesautonomic, and sensory control into the spinal cord,43-45

is near to and influenced by the suprachiasmatic nucleus.Qu et al.45 reported that the diencephalic A11 dopami-nergic neurons in rodents possess long axons extendingover several segments, possibly traversing the entirelength of the spinal cord. Both A10 and A11 group

RLS ANIMAL STUDIES S461


dopaminergic neurons project into the spinal cord inmice, although A11 predominate.46

The first published approach to create an animal modelof RLS was performed by Ondo et al.47 In a stereotaxi-cally operation 6-OHDA was injected bilaterally in thediencephalic A11 nuclei of rats. Histology revealed thatthis caused a 50% reduction of tyrosine hydroxylase(TH)-positive cells in the A11 region. Interestingly, theA11 cells proved more resistant to 6-OHDA lesioningthan the dopaminergic cells of the substantia nigra andthe ventral tegmental area. These, however, were rela-tively preserved. The behavior of lesioned and sham-operated rats was observed for several months.Compared to controls, the 6-OHDA-lesioned rats dem-onstrated a longer latency to sleep, less sleep time (bothassessed by visual observation), and more episodes ofstanding upright during the observation periods. Subse-quently, pramipexole-treated rats showed less standingepisodes and total standing time when compared withuntreated lesioned rats. In this study no sleep recordingswere performed and no movements observed that overtlyresembled human PLMS.

In a subsequent study, A11 lesions were combinedwith iron deprivation.48 Half of the mice received aregular diet and the other half were fed a low-iron diet.One month after dietary manipulation, half of the dietarysegregated group and half of the sham group receivedbilateral 6-OHDA lesions of the A11 nuclei. Post mor-tem histology demonstrated a marked reduction in A11TH staining cells in mice injected with 6-OHDA. Ironlevels in serum, brain, and spinal cord were significantlydecreased after iron deprivation. Interestingly, CNSstores of iron were more reduced in the animals that hadA11 lesioning than those without lesioning, despite sim-ilar serum iron levels. Iron was also much more reducedin the spinal cord, compared to the brain or serum.46

Locomotor activity was significantly increased in bothiron deprived mice and A11 lesioned mice compared tocontrols. The combination of iron deprivation and A11lesioning further augmented motor activity. In addition,the mice in the combined lesioned iron deprivation groupwere more aggressive. Compared to controls, the in-creased activity in A11 lesion mice with or without irondeprivation returned to baseline after treatment with theD2/D3 agonist ropinirole, but was actually increased bythe D1 agonist SKF38393. The D2 antagonist haloperi-dol also worsened the locomotor activity in general, butnot specifically in the lesioned or iron deprived animals.

However, in this study sleep was not assessed. Al-though the combined iron deprivation and A11-lesioninginduces some features of human RLS, an increase inmotor activity per se is quite unspecific. In further ex-

periments an extended behavioral characterization willhave to be done.

D3-Receptor-Hypothesis

While the usually good therapeutic response to dopa-mine and dopamine agonists allows to speculate on aninvolvement of dopaminergic pathways in the pathogen-esis of RLS, nothing is known on the role of differentdopamine receptor subtypes. Recently, focus has beenput on the D3-receptor-mediated transmission, as possi-bly disturbed in RLS. Although D3-receptors are pre-dominantely found in limbic areas of the brain, they arealso present in sensorimotor pathways49,50 and in thespinal cord.51 Interestingly, the diencephalospinal dopa-minergic (A11) fibers project mainly to the dorsal horn,where spinal D3-receptor density is the highest.51 In RLSpatients, excitability of spinal reflexes can be (state-dependently) facilitated,9 an observation that also can bemade in D3-receptor knock-out mice.52 These mice showa phenotype with increased locomotor activity.53 Alsopharmacological studies with preferential D3-receptor-antagonists found increased spontaneous locomotion54,55

and reduced sleep56 in rodents. In contrast, preferentialstimulation of D3 receptor sites inhibited spontaneouslocomotion in healthy rodents in previous studies.49,56,57

Only one study was performed with the aim to char-acterize animals with respect to circadian motor activityand sleep after selective dopamine D3-antagonism: Re-peated treatment with the highly selective dopamine-antagonist SB-277011,58 in contrast to sulpiride (D2/D3antagonist) and placebo, led to an increased sleep latencyafter 2 weeks of treatment. In this study no significantquantitative effect on circadian locomotor activity wasobserved. However, a qualitative change in exploratorybehavior with an increase in center time was observedonly after treatment with the selective D3-antagonist.59

Although the study was designed to screen for behavioralchanges that would be expected in an animal model ofRLS, D3-antagonism induced only some aspects of RLS.A study investigating the occurrence of PLM in a simi-larly treated group is presently being conducted.

Although experimentally induced dysfunction of D3mediated pathways induces some features of RLS inanimals, so far no complete RLS-phenotype has beendocumented after experimental manipulation of thissystem.

Observation of Periodic Limb Movements inAnimals

Periodic limb movements (PLM) are periodically ap-pearing involuntary movements of the lower limbs dur-ing wakefulness (PLMW) or sleep (PLMS).60 PLMS can



occur as an isolated phenomenon but are frequentlyassociated with RLS, and are a symptom of this disorderthat can be measured electrophysiologically.

Okura et al.61 reported the spontaneous occurrence ofbilateral slow leg movements in the sleep of four hypo-cretin-receptor-2 mutant Dobermans, a canine model ofnarcolepsy. The authors described these movements aslasting for 0.5 to 1.5 seconds and occurring repeatedly(4–20 times) at intervals of 3 to 20 seconds, thus havingconsiderable similarities to human PLMS. Furthermorethey mentioned, without giving further details, that theadministration of D2/D3 antagonists increased the inten-sity and severity of those movements. The report islimited by the lack of quantification and by the fact thatthere was no control group.

In an attempt to systematically observe whether PLMSor similar phenomena can be found in rodents, Baier etal. performed polysomnographies in groups of young andold rats.62 In addition to sleep recordings with EEG andneck muscle EMG, movements of the hindlimbs in Non-REM -sleep were registered with a magneto-inductivedevice. Sleep stages and periodic hindlimb movements insleep (PHLM) were scored according to criteria similarto those applied in human sleep recordings. From bothnormal adults and RLS patients, it is known that theoccurrence and number of PLM increase with age. In-terestingly, only animals in the group of aged rats spon-taneously displayed limb movements in Non-REM-sleep, which fulfilled the criteria for PLMS. Only a fewlimb movements in Non-REM-sleep and no PHLM wereobserved in the group of young rats. The dopamine-antagonist haloperidol failed to quantitatively aggravatethe PLM in the observed animals. However, in the agedanimals spontaneously displaying high numbers of limbmovements in NREM sleep (n � 3), haloperidol signif-icantly reduced the intermovement intervals (Baier et al.,unpublished data). To validate whether the observedPHLM are a phenomenon comparable to human PLMS,the response to pharmacological treatments needs to bebetter characterized in a larger number of PHLM positiveanimals.

Recently, Baier et al.63 and Manconi et al.64 indepen-dently performed chronic recordings of tibialis anteriormuscle EMG in parallel to sleep recordings in freelymoving rats. Both found that in young animals, no limbmuscle activity could be seen in NR-sleep, whereastwitches occurred in REM-sleep. In the future, it will benecessary to establish possible and reliable PLM scoringcriteria for these animals.

Lai and Siegel65 found coordinated rhythmic limbmovements—sharing similarities with PLM—in decere-brated cats after lesions in the ventral part of the me-

sopontine junction and the retrorubral nucleus (A8), oneof the dopaminergic midbrain nuclei. These results mayindicate that dysfunction of A8 could release motor ac-tivity in sleep and therefore contribute to the occurrenceof PLM in RLS. However, the observations were madein decerebrated animals, and the influence of dopamine-agonistic or -antagonistic treatments was not assessed.

There is one report that spinal cord lesions led to limbmovements during sleep in a high number of lesionedanimals, while the control group showed no limb move-ments during an 8-day sleep-recording period.66 This isin accordance with observations made in humans, whereRLS and PLMS started after spinal cord lesions.40,41 Themajor limitations of this study are that no informationwas provided about the quantity of limb movements andno statement was made whether the limb movementsoccurred periodically. Also, no pharmacological manip-ulations were performed.

PLMs are a phenomenon associated with RLS thathave been observed spontaneously in narcoleptic dogs,aged rats, and in animals after lesions in the A8 andspinal cord. Whether these observations resemble humanPLMS has still to be validated.

CONCLUSIONS

To date the major limitation of modeling RLS inanimals is the fact that there is neither a defined behav-ioral phenotype of this disorder nor a fully understoodpathyphysiological condition leading to this disorder.Only—so far quite nonspecific—surrogate parameterscan be observed based on behavioral and biologicalchanges typical for RLS.

However, on the bases of the current hypotheses onthe pathophysiology of RLS, some promising attemptshave been made to model RLS in animals, by lesioning,dietary manipulations, and pharmacological interven-tions. The challenge of future animal experimental workwill be to closer investigate the present approaches. Thiswill have to include circadian locomotor and sleep as-sessments and the development of tests for the urge tomove. Also, possibly associated features as PLMS,changes in spinal cord excitability, pain thresholds, andbiological markers will have to be investigated. Thenanimal models will provide methods to evaluate multiplefactors contributing to RLS and possibly theirinteraction.

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