Motor evoked potentials in a rhesus macaque modelof neuro-AIDS

Leigh AM Raymond1,2, Dennis Wallace2, Joanne K Marcario1,2, Ravi Raghavan3, Opendra Narayan2,3,Larry L Foresman4, Nancy EJ Berman2,5 and Paul D Cheney*,1,2

1Department of Molecular & Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd., KansasCity, Kansas, KS 66160, USA; 2Smith Mental Retardation and Human Development Research Center, University ofKansas Medical Center, 3901 Rainbow Blvd., Kansas City, Kansas, KS 66160, USA; 3Marion Merrell Dow Laboratory forViral Pathogenesis, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, Kansas, KS 66160, USA;4Laboratory Animal Resources, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, Kansas, KS66160, USA; 5Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, Kansas,KS 66160, USA

Previous work using bone marrow passaged SIVmac239 (simian immunode®-ciency virus) has shown that macrophage tropic strains of this virus enter therhesus macaque brain early following inoculation (Sharma et al, 1992;Desrosiers et al, 1991; Zhu et al, 1995; and Narayan et al, 1997). As part of aneffort to more fully characterize the extent of neurologic impairment associatedwith SIV infection of the brain, we used transcranial electrical stimulation ofmotor cortex and the spinal cord to evoke EMG potentials in two forelimb (EDCand APB) and two hindlimb (LG and AH) muscles. The latencies, magnitudesand thresholds of motor evoked potentials (MEPs) recorded from nine monkeysinfected with neurovirulent SIVmac R71/17E were compared to pre-inoculationrecords from the same monkeys. Seven of nine monkeys developed simianAIDS within 4 months of inoculation and were euthanized. Two monkeysremained free of AIDS-related clinical illness for over 18 months followinginoculation. Six of the seven monkeys with rapidly progressing disease showedpost-inoculation latency increases (52 s.d. of control) in at least one corticalMEP. Increases in cortical MEP latency ranged from 21 ± 97% in differentmonkeys. All seven rapidly progressing animals showed post-inoculationincreases in at least one spinal cord MEP latency. Maximum spinal cord MEPlatency increases ranged from 22 ± 147%. Increases in central conduction time(CCT) ranged up to 204% and exceeded two standard deviations of control infour monkeys. Neither of the two monkeys with slowly progressing diseaseshowed signi®cant increases in either cortical or spinal cord MEP latency orCCT. Only the monkeys with rapidly progressing disease exhibited classicAIDS-related neuropathology, although there was no consistent relationshipbetween the severity of neuropathology and the extent of MEP abnormalities. Inconclusion, our results demonstrate clear de®cits in the functional integrity ofboth central and peripheral motor system structures associated with SIVinfection and further support the use of SIV-infected rhesus macaques as amodel of neuro-AIDS.

Keywords: monkey; SIV; HIV; AIDS; MEPs; motor de®cits; nervous system

Introduction

Neurological complications are a common ®ndingin patients with symptomatic HIV-1 (human im-munode®ciency virus) infection. About 10% of

AIDS patients ®rst present with a neurologicalcomplaint (Janssen, 1997). HIV-1 associated demen-tia occurs in approximately 20% of patients and ischaracterized by (1) cognitive impairment includingmental slowness, forgetfulness and poor concentra-tion; (2) behavioral changes including apathy,lethargy as well as diminished, dysphoric orinappropriate emotional responses; and (3) motorsymptoms including the loss of ®ne motor control,

*Correspondence: PD Cheney, Smith Mental Retardation ResearchCenter, University of Kansas Medical Center, 3901 Rainbow Bou-levard, Kansas City, KS 66160, USAReceived 9 July 1998; revised 21 December 1998; accepted 25January 1999

Journal of NeuroVirology (1999) 4, 217 ± 231

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1999 Journal of NeuroVirology, Inc.

unsteady gait and tremor (Navia et al, 1986a,b;Navia and Price, 1987; Saykin et al, 1991; Glass andJohnson, 1996; Dal Pan et al, 1997).

It has been known for many years that HIV-1enters the central nervous system (CNS) earlyfollowing exposure (Michaels et al, 1988; Sharer,1992; Epstein and Gendelman, 1993). Once withinthe CNS, HIV-1 preferentially infects microglia(CNS resident macrophages), although virus hasalso been isolated from astrocytes (Takahashi et al,1996). Primary infection of neurons has not beenconvincingly demonstrated (Glass and Johnson,1996). Neuronal loss associated with HIV-1 infec-tion of the CNS, therefore, has been attributed tosecondary processes (Everall et al, 1993, 1994; Glassand Johnson, 1996), although some dispute theoccurrence of neuron loss (Seilhean et al, 1993).Increasing evidence points to several potentialneurotoxic factors including cytokines, chemo-kines, excess neurotransmitters and reactive oxygenspecies that may be released by HIV-1 infectedmacrophages or astrocytes and may trigger anexcitotoxic cascade (Lipton, 1997).

Motor system de®cits are a common componentof HIV-1 related cognitive/motor disorder andAIDS-related dementia (Glass and Johnson, 1996;Dal Pan et al, 1997). Motor evoked potentials (MEPs)have been used to investigate the integrity of bothperipheral and central motor pathways in a numberof human disease conditions including HIV-1infection (Arendt et al, 1992; Moglia et al, 1991;Connolly et al, 1995). MEPs are electromyographic(EMG) potentials recorded from muscles in re-sponse to either electrical or magnetic stimulationof the cerebral motor cortex or the spinal cord.Stimulation of motor cortex provides a measure ofconduction time along both central corticospinalpathways to spinal cord motoneurons and periph-eral conduction from the spinal cord to muscles.Hence, cortical motor evoked potentials provide ameasure of total conduction time. Stimulating thespinal cord, on the other hand, provides a measureof conduction time along just the peripheral part ofthe pathway. A measure of central conduction timecan be obtained by subtracting spinal cord MEPlatency from cortical MEP latency. Based on a studyof 138 HIV-infected patients, Moglia et al (1991)reported signi®cant increases in central conductiontime in 44% of asymptomatic HIV positive patientsand 72% of patients with AIDS. Several studieshave also reported slowing in peripheral motorconduction time in both asymptomatic and sympto-matic HIV-infected patients (Cornblath andMcArthur, 1988; Jakobsen et al, 1989; Fuller et al,1991), although the presence and extent of MEPabnormalities in HIV-infected, asymptomatic indi-viduals has been disputed (McAllister et al, 1992).

Animal models offer important advantages for thestudy of retroviral neuropathogenesis. By passagingSIVmac239 through bone marrow, Narayan and

colleagues developed highly neurovirulent strainsof macrophage-tropic SIVmac (Sharma et al, 1992;Narayan et al, 1997). As in HIV-1 disease, SIV isknown to enter the brain early following inocula-tion and cause productive infection of microglia,and possibly astrocytes, but not neurons (Narayan etal, 1997). As in HIV-1 disease, neuronal loss hasalso been reported in SIV-infected macaques, bothin the brainstem and in the cortex (Berman et al,1998; Adamson et al, 1996; Weihe et al, 1993).

Prospero-Garcia et al (1996) demonstrated neuro-physiological alterations in cortical and brainstemresponses to visual and auditory stimuli in rhesusmacaques infected with microglial-passagedSIVmac251. More recently, Raymond et al (1998) re-ported delays in the latencies of peaks in the auditorybrainstem response of SIV-infected macaques.

Behavioral performance in SIV-infected monkeyshas also been examined. Murray et al (1992) ®rstdemonstrated cognitive and motor impairments inSIV-infected rhesus monkeys using the Delta B670strain of SIV. Although cognitive de®cits wereobserved in some monkeys on delayed match tosample and visual discrimination and learningtests, nearly all of the monkeys tested (8/10) wereimpaired on a test of motor skill requiring themonkey to retrieve a food pellet from a rotating disk.More recently, Fox et al (1997) reported that rhesusmacaques infected with microglial-associated SIVshowed de®cits in attention set shifting, bimanualmotor skill and progressive ratio task performance.We have found that monkeys infected with the sameneurovirulent strains of SIV used in the presentstudy showed clear de®cits in reaction time andmovement time on simple and choice reaction timetasks (Marcario et al, 1999). However, decisionmaking time (choice minus simple reaction time)was not consistently affected. Taken together, thesestudies suggest that while de®cits in cognitivefunction occur in association with SIV diseaseprogression, motor system dysfunction may bemore prominent.

Previous studies in both HIV-1 infected humansand SIV-infected monkeys have demonstratedneuronal loss in motor cortex suggesting that thecorticospinal system is a target of damage (Masliahet al, 1992; Everall et al, 1993; Weihe et al, 1993).Therefore, the purpose of this study was to test thefunctional integrity of the forelimb and hindlimbmotor output apparatus in rhesus macaques inocu-lated with a combination of two neurovirulentstrains of SIVmac. Our ®ndings show signi®cantslowing in spinal cord (peripheral conduction time)and cortical motor evoked potentials (MEPs) inmonkeys with rapidly progressing disease, occur-ring in as little as 6 weeks following inoculation.Subtracting spinal cord MEP latency from corticalMEP latency revealed that central conduction timewas also increased in four of seven monkeys withrapidly progressing disease. Neither of two mon-

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keys with slowly progressing disease showedincreases in peripheral or central conduction time.Demonstration of SIV-induced motor system patho-physiology con®rms the appropriateness of thismodel for further investigations of the mechanismof AIDS-related neurological impairment.

Results

Mortality and morbidityA total of nine Rhesus macaques were inoculatedwith SIVmacR71/17E via femoral bone marrowinjection at two different time-points. Four of thenine animals served initially as controls, but after 6months these monkeys were also inoculated toincrease sample size. Seven of nine monkeysprogressed to end-stage AIDS within 16 weeks ofinoculation and were considered rapid progressors.At necropsy, monkeys exhibited one or more of thefollowing clinical signs: weight loss, diarrhea,tremor, ataxia, weakness, dermatitis, gingivitis, orallesions, skin lesions, and dysphagia (Table 1). Oneanimal (AQ69) died prematurely from anesthesiaassociated with a MRI. At the time of death, thismonkey had mild weakness and weight loss. Twoanimals became productively infected with SIV butsurvived for 109 (AQ15) and 87 weeks (AQ94)weeks and were considered slow progressors. At thetime of euthanasia, one of these monkeys (AQ15)exhibited widely disseminated skin tumors, mildwasting and anemia, possibly due to persistentepistaxis; the other monkey (AQ94) was jaundiceand showed signs consistent with acute liverdisease.

Reproducibility of MEPsFigure 1 shows examples of single trial EMGresponses from two muscles elicited by electrically

stimulating the motor cortex (cortical MEPs) andspinal cord (cervical cord MEPs) at differentintensities (1.25, 1.5 or 3.0 times threshold for amotor response). Averages of the single trials shownin each column are given in the bottom row of the®gure. All measurements were made from averagedrecords. The somewhat greater trial-to-trial varia-bility in cortical MEPs compared to spinal cordMEPs is probably due largely to ¯uctuations incortical and motoneuronal excitability. Neverthe-less, latency and magnitude variability in bothspinal and cortical MEPs were relatively smallyielding average records with highly reproduciblefeatures.

Cortical MEP latency changesOf the seven animals that progressed rapidly to end-stage disease, ®ve (AQ70, 12, 43, 38, 47) showedpost-inoculation increases in cortical hindlimbMEP latency that exceeded the control mean bymore than two standard deviations (Table 2).Increases were present for both the lateral gastro-cnemius (LG) and abductor hallucis (AH) muscles.Six of seven monkeys (all except AQ12) showedincreases in cortical forelimb MEPs, although onlytwo of these monkeys (AQ47, AQ38) had increasesin both extensor digitorum communis (EDC) andabductor pollicis brevis (APB) muscles. Combiningforelimb and hindlimb cortical MEPs, one or morelatency was increased in all seven monkeys. Fourmonkeys showed increases in three or more corticalMEPs; two showed increases in all four corticalMEPs.

Figure 2 shows the distribution of latencychanges in SIV-infected monkeys obtained duringthe ®nal MEP session (end-stage AIDS) compared topre-inoculation latencies. Data for control monkeysare based on MEPs obtained over a comparable

Table 1. Clinical signs of SIV infected monkeys.

MonkeyDisease

progression Inoculated DeathWeeks

survived Clinical signs at necropsy

AQ70 Rapid 01/12/96 04/18/96 14 Weight loss, facial and generalized subcutaneousedema, ataxia, skin lesions, loss of appetite, weakness

AQ69 Rapid 01/12/96 03/16/96 9 Weight loss, resting tremor, epistaxis, skin lesions, weaknessAQ43 Rapid 01/12/96 02/23/96 6 Weight loss, diarrhea, cyanosis, severe ataxia, dysphagia,

bleeding gumsAQ12 Rapid 01/12/96 04/16/96 14 Weight loss, diarrhea, cyanosis, severe tremor, severe ataxia,

weaknessAQ47 Rapid 07/03/96 08/27/96 8 Weight loss, diarrhea, cyanosis, slight

tremor, minor ataxia, loss of appetiteAQ38 Rapid 07/03/96 08/23/96 7 Weight loss, diarrhea, tremor, severe ataxia, skin rash,

loss of appetite, weaknessAQ20 Rapid 07/03/96 08/26/96 8 Weight loss, diarrhea, cyanosis, ecchymosis, oral lesionsAQ15 Slow 01/12/96 02/10/98 109 Weight loss, cyanosis, dermatitis, diarrhea, edema, epistaxis,

oral lesions, skin lesions, tumorsAQ94 Slow 07/03/96 03/04/98 87 Weight loss, cyanosis, jaundice, diarrhea, weakness, loss of

appetite

N/A=not available.

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219

period of time (6 months). The results presented inthis ®gure differ slightly from the summary in Table2 because Table 2 takes into account data from allMEP recording sessions. As in Table 2, this ®gure,depicting changes for the ®nal MEP session, showsthat all rapidly progressing monkeys had at leastone MEP latency increase that exceeded twostandard deviations. None of the latencies of theslowly progressing monkeys exceeded two standard

deviations. Maximal increases in cortical MEPlatency ranged from 21 ± 97% in different rapidlyprogressing animals. Changes in cortical MEPs forcontrol monkeys were centered about zero andranged in magnitude from 77.6 to 7.8%. Reduc-tions in latency (negative changes) observed for SIVmonkeys were in the same range as those observedfor control monkeys and can be attributed to normalvariability. Mean latency increases in LG were

Table 2 Summary of MEP latency increases.

Cortical Cord

MonkeyDisease

progressionSIV neuro-pathology{

SIV p27(pg/ml){{

CD4(cells/ml){ EDC APB LG AH

CervicalEDC

CervicalAPB

LumbarLG

LumbarAH

AQ69AQ70AQ12AQ43AQ38AQ47AQ20AQ15AQ94

RapidRapidRapidRapidRapidRapidRapidSlowSlow

N/AMild

ModerateSevereSevereMildMildMildNone

1288383047374902523856455688225

ND{{{

9652356658518

11871540

51177119

**

***

***

*****

*****

******

*

*

**

******

*

***

N/A=not available, ND=not detectable. {Applies to motor system structures: motor cortex, basal ganglia, cerebral peduncles, longwhite tracts, and peripheral nerve. *Latency increase 52 s.d. of the mean. {{SIV serum p27 is a measure of viral load. {{{AQ94 hadp27 protein levels as high as 332 earlier during the study. {The normal range for CD4 cell counts is 1000 ± 2000/ml. Both p27 and CD4cell counts are based on the ®nal measurement prior to euthanasia.

Figure 1 Examples of cortical and cervical cord MEPs illustrating how the data was collected. Ten single trials were collected forcortical MEPs at 1.25 and 1.5 times threshold. For spinal cord MEPs, ®ve single trial records were collected at 1.25 and 1.56threshold;two to three were collected at 36threshold. The single trial records were then full-wave recti®ed and averaged. MEP amplitude for the36 threshold average record was considered the maximum MEP and assigned a value of 100% for the purpose of quantifying all otherrecords obtained for that muscle.

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somewhat greater than those in AH (20% versus8%); mean increases in EDC were greater than thosein APB (36% versus 7%).

Figure 3 shows examples of cortical MEPs fromfour different monkeys. Records for two rapidlyprogressing SIV-infected monkeys exhibit clearlatency increases while those for a control monkeyand a slowly progressing SIV-infected monkeyshow no changes over a similar period of time.

Spinal cord MEP latency changesSix of the seven SIV-infected monkeys with rapidprogressing disease (all except AQ69) showed post-inoculation increases in spinal cord hindlimb MEPlatencies that exceeded the control mean by morethan two standard deviations (Table 2). Four ofthese six showed increases in both hindlimbmuscles tested. All seven monkeys showed in-creases in spinal cord forelimb MEPs, althoughboth EDC and APB latencies were increased in onlythree of these cases. Neither of the two monkeyswith slowly progressing disease showed increasesin spinal cord MEP latencies.

Maximum cord MEP latency increases for differ-ent rapidly progressing SIV-infected monkeysranged from 33 ± 147% (Figure 2). Changes forcontrol monkeys were centred about zero and

ranged in magnitude from 79.3 to 13.8% fordifferent animals. As with cortical MEPs, latencyincreases in LG tended to be greater than those inAH (mean increase of 62% versus 43%), and EDCappeared to be more strongly affected than APB(mean increase of 25% versus 11%).

Figure 4 shows examples of spinal cord MEPsfrom four different animals. Records for twodifferent rapidly progressing SIV-infected monkeysexhibit clear latency increases while those for acontrol monkey and a slowly progressing SIV-infected monkey show no changes over a similarperiod of time.

Central conduction time (CCT) changesAs illustrated in Figure 5, cortical MEP latency is ameasure of total conduction time and represents thesum of peripheral (spinal cord MEP latency) andcentral conduction time. Total conduction time isthe sum of conduction along corticospinal axons tospinal motoneurons, synaptic transmission at themotoneuron, time to bring the motoneurons to ®ringthreshold, peripheral nerve conduction, neuromus-cular transmission and conduction along muscle®bers to the recording electrodes (Figure 6). Spinalcord MEP latency is a measure of the peripheralcomponent of this pathway including conductionalong peripheral motor axons, neuromusculartransmission, and conduction along muscle ®bersto the EMG electrode recording site. CCT isroutinely computed by subtracting peripheral con-duction time (spinal cord MEP latency) from thecorresponding total conduction time (cortical MEPlatency).

We calculated CCTs for our SIV-infected mon-keys and for data collected from the four animalsthat initially served as controls. Control data wereused to de®ne the two standard deviation bound-aries. Changes in CCT were examined by calculat-ing the per cent change in post-inoculation CCT(based on data from the ®nal MEP session)compared to pre-inoculation CCT for each mon-key. CCT data for control animals was collectedover the same length of time as that for the rapidprogressors. Four of the rapid progressors (AQ20,AQ12, AQ38, AQ47) showed 50% or greaterincreases in CCT, well above the two standarddeviation criterion (Figure 7). Some smallerdecreases in CCT were also observed, althoughmechanisms other than increased conductionspeed probably explain these changes (see Discus-sion). The two monkeys with slowly progressingdisease did not show changes in CCT that differedsigni®cantly from changes observed in controlmonkeys. Although individual monkeys showedincreased CCTs, the mean change in CCT for allSIV-infected monkeys of 13.8+23.9% (s.d.) wasnot signi®cantly different (P40.3, Mann-Whitneyrank sum test) from the increase of 3.15+13.6%(s.d.) observed in control animals.

Figure 2 Distribution of cortical and spinal cord MEP latencychanges in all nine SIV-infected monkeys and four controlmonkeys. The dashed lines represent+2 s.d. from the mean ofcontrol monkeys. Individual points correspond to MEP latenciesfor different muscles. Each column contains eight points but insome cases the points are overlapping. Asterisks indicate meanvalues. Note that the data in this plot differ slightly from Table 2because this plot is based on the ®nal post-inoculation MEPsession whereas the statistical analysis for Table 2 took intoaccount data from all post-inoculation recording sessions. Someof the SIV and control monkey numbers are the same becausefour monkeys were used over a period of 6 months to collectcontrol data. They were then inoculated to increase the size ofthe SIV group.

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Cortical MEP magnitude and threshold changesChanges in the magnitude of cortical MEPs wereexpressed as a percentage of maximal (36thresh-old) spinal cord MEP magnitude. Pre-inoculationcortical MEP magnitudes were similar for theforelimb and hindlimb and ranged from 4 ± 60% ofmaximum in different muscles. Post-inoculationmagnitudes ranged from 1 ± 52%. SIV monkeysshowed no signi®cant changes in cortical MEPmagnitude over a similar time period as that usedfor control monkeys.

Over the course of the study, we also measuredstimulus thresholds for evoking cortical and spinalcord MEPs. Threshold was expressed as a percen-tage of maximum stimulator output. Pre-inoculationmean thresholds for the SIV-infected monkeys did

not differ from the mean thresholds of the last post-inoculation MEP session. Pre-inoculation meancortical hindlimb and forelimb MEP thresholds were24+7% (s.d.) and 20+6% respectively; the corre-sponding post-inoculation thresholds were 23+5and 20+8%. Pre-inoculation mean spinal cordhindlimb and forelimb MEP thresholds were 17+3and 20+4% respectively; the corresponding post-inoculation thresholds were 19+4 and 21+7%.

Onset of conduction delays in relation to diseaseprogressionIn all cases except two, signi®cant MEP conductiondelays were only observed for the last evokedpotential series when the monkey had reachedend-stage disease. Two monkeys (AQ12 and AQ70)

Figure 3 Examples of cortical MEPs recorded over a period of 4 ± 5 months in SIV-infected monkeys and a control monkey. Panels Aand B show records for two SIV-infected monkeys that progressed rapidly to end-stage disease. The last record shown was the ®nalMEP obtained and corresponded with end-stage disease. Panel C shows records for an uninoculated control monkey. Panel D showsrecords for a slowly progressing SIV-infected monkey that remained free of clinical signs over the same period of time as data shownfor the other monkeys. Cortical MEP latencies did not increase in either the control or slowly progressing monkeys. Light gray recordswere obtained after the date of inoculation (1/12/96).

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showed conduction delays in two sessions prior tonecropsy providing evidence for progression ofneurologic disease. Monkey AQ12 showed progres-sive increases in cortical and spinal cord hindlimbMEP latency for two sessions prior to necropsy(Figure 3A); monkey AQ70 showed increases inlumbar spinal cord MEPs for two sessions prior tonecropsy (Figure 4A). Spinal cord latency changeswere observed 2 weeks prior to increases in latencyof cortical responses for both AQ12 and AQ70.

Other signs of motor system pathophysiologySix rapid progressors (all except AQ20) exhibitedclinical signs of motor system pathophysiologyincluding ataxia and tremor. One of these monkeys(AQ12) also showed enhanced stretch re¯exes andclonus of the ankle extensors (Figure 8). Corticaland spinal cord MEP latency increases were alsoobserved. In fact, this monkey showed one of thelargest increases in overall post-inoculation MEPlatency (Figure 2).

Figure 4 Examples of spinal cord MEPs obtained over a period of 4 ± 6 months in SIV-infected and a control monkeys. Panels A andB show records for two SIV-infected monkeys that progressed rapidly to end-stage disease. The last record shown was the ®nal MEPobtained and corresponded with end-stage disease. Panel C shows records for an uninoculated control monkey. Panel D shows recordsfor a slowly progressing SIV-infected monkey that remained free of clinical signs over the same period of time as data shown for theother monkeys. Spinal cord MEP latencies did not increase in either the control or slowly progressing monkeys. All monkeys wereinfected on 1/12/96. Light gray records were obtained after inoculation.

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Neuropathological, virological and immunologicalcorrelationsEvidence of typical retroviral induced CNS neuro-pathology was observed in seven of eight animalsfrom which tissue was available. Neuropathological®ndings consisted of perivascular cuf®ng, micro-glial nodule formation, multi-nucleate giant cellreactions and peri-lesional axonal degenerationcaused by white matter in¯ammatory lesions. Theseverity of pathology varied from animal to animal.Macaques AQ43 and AQ38 had severe disseminatedmeningo-encephalomyelitis in the form of nodularand perivascular mononuclear in¯ammatory cellin®ltrates composed of monocyte-macrophages andmicroglia, multinucleate giant cells (MGCs) andsome lymphocytes (Table 2). In white matter, thesein®ltrates caused mild perilesional axonal damageand demyelination. Systematic examination of thedistribution of lesions along the central andperipheral motor pathways revealed extensiveinvolvement of not only the long tracts (internalcapsules, cerebral peduncles, pyramids and des-cending spinal tracts), but also the grey matter of themotor cortex and deep nuclei. This was accompa-nied by focal neuronal destruction in the affected

areas. In contrast, macaques AQ12, AQ70, AQ47and AQ20 demonstrated lesions of less intensityand dissemination. Pathology in these animals wasrestricted to the cerebral white matter and longtracts of the central motor pathways. Grey matterchanges or obvious neuronal destruction was not afeature in these animals.

Signi®cant involvement of the spinal anteriorhorn cells or peripheral nerves was not obvious. Atnecropsy, samples of peripheral nerve (median,tibial, sciatic and femoral) were routinely taken andexamined for evidence of pathology using lightmicroscopy and tissue sections stained with hema-toxylin and eosin. Peripheral nerve lesions wererare and seen only in macaque AQ12.

It is noteworthy that all monkeys with rapidlyprogressing disease, in which a pathological analy-sis was possible, showed classic AIDS-relatedneuropathology (microglial nodules and multi-nucleate giant cells). In contrast, neither of the twoanimals with slowly progressing disease showedclassic AIDS-related neuropathology, even thoughthe post-inoculation life-spans of these monkeyswere much longer and they did develop clinicalAIDS-related disease. AQ15 showed only mild,

Figure 5 Measurement of MEP latencies. Peripheral conduction time was measured from stimulation of the cervical or lumbar spinalcord. A measure of total conduction time (peripheral and central) was obtained from transcranial electrical stimulation of motorcortex. An estimate of central conduction time (CCT) was obtained by subtracting peripheral conduction time from total conductiontime.

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focal meningo-encephalitis and AQ94 had noneuropathological changes involving motor systemstructures. However, it should be noted that theseverity of motor system neuropathology did notalways match the extent of MEP abnormalities. Forexample, AQ47 and AQ70 had only mild neuro-pathological changes, but both of these monkeysshowed marked changes in nearly all MEPs tested(Table 2, Figure 2).

Neither was there a consistent relationshipbetween viral load (p27 levels) or CD4 cell countand the extent of MEP abnormalities within thegroup of rapid progressors (Table 2, Figure 2).However, the fact that the slow progressors showedno MEP de®cits and had very low viral loads andalso low CD4 cell counts compared to the rapidprogressors suggests that viral load is a moreimportant predictor of neurological injury.

Discussion

In this study we present evidence for delays incortical and spinal cord MEPs associated withneurovirulent SIV infection in rhesus macaques.Of nine infected monkeys on which data was

Figure 6 Illustration of the motor output pathways underlying cortical and spinal cord MEPs. Transcranial electrical stimulationevokes a descending volley of action potentials in corticospinal neurons (CSN). Many of these neurons make monosynaptic excitatoryconnections with large and small alpha motoneurons. However, at the relatively low intensities of stimulation used in this study,cortical MEPs will primarily re¯ect activation of the smaller motoneurons due to the principle of orderly recruitment according to size.In contrast, because electrical stimulation activates large axons at lower stimulus intensities then small axons, our spinal cord MEPswill primarily re¯ect activation of large motor axons. This has important implications for interpreting central conduction time data.

Figure 7 Distribution of central conduction time (CCT) changesfor all nine SIV-infected monkeys and four monkeys thatinitially served as control animals. The dashed lines represent+2 s.d. from the mean of the control data. Individual pointsillustrate CCT for different muscles. Each column contains fourpoints but in some cases the points are overlapping. This plot isbased on the per cent difference between the pre-inoculationand ®nal post-inoculation CCTs for each animal and muscle.Control data was collected over a period of 6 months and the percent difference between the ®rst and last control sessions areshown.

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collected, seven progressed to end-stage diseasewithin a period of 4 months or less (rapidprogressors) and all of these monkeys showedlatency increases in at least one of eight MEP types(cortical and spinal cord MEPs for each of fourmuscles). In some cases, latency increases werepresent over multiple post-inoculation test sessions.However, the magnitude of latency increases weremost prominent for the last recording sessioncorresponding to end-stage disease.

In two monkeys (AQ15 and AQ94) diseaseprogression was slow. At 87 and 109 weeksrespectively, complications related to systemicdisease (wasting, liver dysfunction and skin tumors)necessitated euthanasia. At necropsy, these twoanimals had p27 levels that were much lower thanmonkeys with rapidly progressing disease (Table 2).In fact, blood levels of p27 remained low (560 pg/ml) over most of the disease course. CD4+ cellsshowed a progressive decline over a period ofseveral months and at the time of necropsy werelower than most of the monkeys with rapidlyprogressing disease. Neither AQ15 nor AQ94

showed signi®cant MEP latency increases. Neitherdid these two monkeys show increases in thelatency of auditory brainstem responses (Raymondet al, 1998). Based on these ®ndings and thecomplimentary ®ndings of Westmoreland et al(1998), we conclude that these two monkeysdeveloped systemic AIDS in the absence of sig-ni®cant neurological disease and that SIV inducedencephalitis and neurologic impairment is char-acteristic of rapid disease progression.

Spinal cord MEPs provide a measure of periph-eral conduction time (Mills and Murray, 1986;Kimura, 1989). Increased peripheral conductiontime is most easily attributed to axonal conductionslowing or failure, possibly related to demyelina-tion or axonal loss. Despite the fact that peripheralconduction latencies showed large increases (asmuch as 147%), peripheral nerve pathology visibleat the light microscopic level appeared to beminimal. Only one monkey (AQ12) showed clearevidence of peripheral nerve in¯ammatory lesions.However, our histological technique could havemissed more subtle axonal loss or demyelination.Also, peripheral nerve pathology may have beenlocalized to regions of the nerve that we did notexamine. Using simple light microscopy, we ex-amined 2 cm long sections of different peripheralnerves (median, sciatic, posterior tibial, and femor-al) in both longitudinal and transverse sections. Inthe absence of overt structural pathology, conduc-tion slowing could result from a pathophysiologicalprocess affecting ion channels and excitability(Koller et al, 1997). In any case, the increases inspinal cord MEP latencies indicate the presence ofunderlying peripheral nerve pathophysiology if notactual neuropathy or myelopathy.

We computed CCT by subtracting spinal cordMEP latency (peripheral conduction time) from thecorresponding cortical MEP latency (total conduc-tion time). We observed substantial increases inCCT in four monkeys suggesting slowing of centralmotor pathways. This is consistent with clearevidence of CNS white matter pathology in ouranimals. It is of interest that changes in CCT wereonly observed for animals with rapidly progressingSIV disease. Monkeys with slowly progressingdisease did not show changes in either central orperipheral conduction time.

It is puzzling that decreases in CCT time werealso observed (Figure 7). The possibility that thismight be due to actual increased central conductionvelocity seems highly unlikely, but there are otherfactors that might explain this result. For example,it is likely that cortical and spinal cord MEPs aremediated predominately by small (slowly conduct-ing) and large (rapidly conducting) motoneuronsrespectively. This is because cortical MEPs involvesynaptic activation of motoneurons whereas spinalcord MEPs involve electrical stimulation of moto-neuron axons (Kimura, 1989). What is most relevant

Figure 8 Abnormal stretch re¯ex response obtained from thelateral gastrocnemius in one SIV-infected monkey (AQ12) thatexhibited clinical neurological symptoms including ataxia andtremor. Rapid ankle ¯exion denoted by the lower recordproduced a re¯ex response that was larger and prolongedcompared to that obtained from a control monkey (B) undersimilar conditions. Even more abnormal was the existence of asecond, equally large re¯ex response suggesting the presence ofclonus. Average records based on 21 trials in A and 14 trials in B.Magnitude was quanti®ed in relation to the 36threshold lumbarcord MEP taken as 100%. Note the difference in vertical scales.

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for our study is the possibility that viral injury maypreferentially affect large axons as suggested byFuller et al (1991). In this case, and given that thecortical and spinal cord MEPs rely on slow and fastconducting motoneurons respectively, marked per-ipheral MEP latency increases could occur withoutcomparable cortical MEP latency increases (Figure6). Comparison of pre- and post-inoculation CCTsmight then yield negative changes, falsely suggest-ing faster conduction (Figure 7).

Studies of MEPs in asymptomatic and sympto-matic HIV-infected humans have yielded con¯ict-ing results. All studies seem to agree that the latestages of disease (CDC stage IV) are characterized bya clear peripheral neuropathy involving bothsensory and motor axons (Cornblath and McArthur,1988), although some even question this (McAllisteret al, 1992). However, several studies reportperipheral conduction slowing associated withearlier, asymptomatic stages of disease (Jakobsenet al, 1989; Fuller et al, 1991), while others ®nd thatperipheral conduction slowing is only presentwhen disease has progressed to a late stage andclinical symptoms are present (Farnarier andSomma Mauvais, 1990; Ronchi et al, 1992). Reportsof central motor slowing are also con¯icting. Mogliaet al (1991) found slowing of transcranial MEPs inboth HIV positive, asymptomatic patients (44%)and patients with AIDS (72%). They attributed mostof the slowing to defects in central motor conduc-tion time. Others have also reported prolongedcentral motor conduction time in asymptomatic,HIV positive patients (Farnarier and Somma Mau-vais, 1990; Somma Mauvais and Farnarier, 1992). Incontrast, Grapperon et al (1993) and Arendt et al(1992) found no evidence of conduction defects incentral motor pathways in either asymptomatic orsymptomatic patients. Our ®ndings support theview that MEP de®cits are present, but associatedwith the later stages of disease progression. Thesede®cits can be attributed to increases in bothperipheral and central conduction time.

All our animals became productively infectedwith SIVmacR71/17E as demonstrated by the sub-stantial levels of p27 found in blood (Table 2). Itshould be noted that AQ94 had viral p27 proteinlevels as high as 332 pg/ml at one point duringdisease progression although in the ®nal sample, p27was undetectable. Of particular interest is the factthat at the time of necropsy, monkeys with slowlyprogressing disease had very low levels of virus butalso had very low CD4 cell counts compared tomonkeys with rapidly progressing disease. Relatingthis ®nding to the fact that slowly progressingmonkeys did not show MEP de®cits, while rapidprogressors did, suggests that viral load is a betterpredictor of neurological injury than CD4 cell count.However, within the group of rapid progressors,neither viral load nor CD4 cell count was a goodpredictor of the severity of MEP abnormalities.

The severity of classic AIDS-related neuropathol-ogy (microglial cells, multi-nucleate giant cells) wasalso variable. Only the monkeys with rapid progres-sing disease developed classic AIDS-related neuro-pathology and only the rapid progressors showedMEP de®cits. Therefore, at this level, there was goodagreement between neuropathological and MEP®ndings. However, it should be emphasized thatwithin the group of rapid progressors, the severity ofpathology was not a good predictor of the severity ofMEP de®cits. For example, two monkeys with onlymild pathology had extensive MEP abnormalities(AQ47 and AQ70; Table 2). Discordance betweenpathology and function has also been noted instudies of HIV-1 infected humans (Glass et al,1993; Everall et al, 1994) and in a previous study ofbehavioural de®cits in rhesus macaques infectedwith the Delta B670 strain of SIV (Rausch et al, 1994).

In conclusion, we have demonstrated substantialpost-inoculation latency increases in both spinalcord and cortical MEPs in SIVmacR71/17E infectedrhesus macaques with rapidly progressing disease.Over half of the monkeys with rapidly progressingdisease also showed substantial increases in CCT.Therefore, both peripheral and central motor path-ways seem to be targets of SIV-related pathophy-siology. We attribute delays in peripheral andcentral conduction to injury of corticospinal neu-rons and motoneurons, respectively. While demye-lination and/or neuronal loss is the most likelyexplanation of this slowing, the lack of obviousperipheral nerve pathology in most of our monkeysraises the possibility of functional injury (Koller etal, 1997). These results further establish theSIVmacR71/17E infected macaque monkey as auseful model of neuro-AIDS, including a possiblemodel for the peripheral myelopathy and neuro-pathy often associated with HIV-1 infection.

Materials and methods

SubjectsNine, adolescent, male rhesus macaques of Indianorigin were inoculated via bone marrow injection ofthe femur with SIVmacR71/17E. Each animal received0.5 ml of each homogenate for a total volume of 1 mlcontaining approximately 1000 TCID50. The mon-keys were free of SIV and Herpes B Virus. Four of themonkeys initially served as sex and age matchedcontrols over a period of 6 months during which datawere also collected from ®ve monkeys infected withSIV. After 6 months, the four control monkeys werealso inoculated to increase the size of the SIV cohort.Daily body temperature and health observationswere noted over the course of disease progression(Table 1). Blood and CSF samples were collectedfrom all animals once a week for the ®rst month afterinoculation, every 2 weeks for the second month,and thereafter at 4-week intervals. All inoculatedmonkeys became productively infected with SIV as

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demonstrated by blood levels of p27. Seven of thenine infected monkeys exhibited rapidly progres-sing disease and were euthanized within 4 months ofinoculation after developing signs of end-stagesimian AIDS. Disease progression was slow in twoother monkeys (AQ15 and AQ94). These monkeyswere euthanized at 109 and 87 weeks post-inocula-tion due to systemic complications including wast-ing, skin tumors and liver disease.

Monkeys were euthanized when one of thefollowing criteria were met: (1) weight loss exceed-ing 20% of body weight, (2) inability to maintain asitting posture, (3) failure to eat or drink, or (4)diarrhea unresponsive to treatment. All proceduresin this study conformed to the Guide for the Careand Use of Laboratory Animals published by theU.S. Department of Health and Human Services,National Institutes of Health. The monkeys in thisstudy were also trained to perform behavioral tasks(reaction time, working memory, and motor skill)that were used to detect cognitive and motorimpairments. A battery of sensory evoked potentialswere also recorded. Results of the behavioural andsensory evoked potential studies will be the subjectof other papers.

MEP recording methodsMEPs were recorded at 4-week intervals includingone, or in some cases, two pre-inoculation sessions.Following an initial injection of ketamine (10 mg/kgwith atropine at 0.1 mg/kg), anesthesia was main-tained by subsequent doses of ketamine (10 mg/kg)every 20 ± 30 min. Body temperature was measuredat the start and completion of each EP session usingan infrared tympanic thermometer. Temperaturewas maintained by surrounding the animal withheated saline bottles during recording. Subdermalplatinum needle electrodes (Grass2 model E2) wereused for differential recording of EMG potentialsevoked by transcranial electrical stimulation of themotor cortex. Recordings were made from twohindlimb muscles ± lateral gastrocnemius (LG) andabductor hallucis (AH), and two forelimb muscles ±extensor digitorum communis (EDC) and abductorpollicis brevis (APB).

MEPs were elicited using a modi®ed bipolar handheld stimulating electrode (Digitimer model DS180-031). The cathode and anode were separated by4 cm and consisted of 1 cm diameter saline soakedpads. Conductive gel was used to further reduceresistance. Current pulses (50 microsecond dura-tion) were generated by a high voltage stimulator(Digitimer2 Model DS180). MEPS were recordedusing Neurolog ampli®ers (Digitimer2 NL820 andNL822 preampli®er) with ®lter settings of 30 Hz ±10 kHz. EMG signals were digitized at 10 KHz andaveraged using custom software (Neural Averager,Larry Shupe, University of Washington, Seattle)written for the Windows2 operating system andCambridge Electronic Design 1401 plus data acqui-

sition hardware. Averaged MEPs were based on tenstimulus repetitions for cortical MEPs and ®verepetitions for spinal cord MEPs.

Cortical MEPs provide a measure of total motorsystem conduction time including central andperipheral components. Cortical MEPs were eli-cited by placing the anode on the scalp overlyingeither hindlimb motor cortex (near Cz) or forelimbmotor cortex (near C3). The cathode was positionedanteriorally on the contralateral side of the skull.MEP thresholds were determined at a gain of 10 Kand expressed as a percentage of maximumstimulator output. The lowest threshold positionwas determined by stimulating at different scalplocations while observing MEPs on an oscilloscopescreen. Cortical MEPs were recorded at 1.25 and 1.5times the threshold of the forelimb or hindlimbmuscle with the highest threshold (Figure 1).

Forelimb and hindlimb spinal cord MEPs weredetermined by positioning the cathode over thecervical and lumbar enlargements respectively.Spinal cord MEPs provided a measure of peripheralmotor system functional integrity. Cord MEPs wererecorded at 1.25, 1.5 and 36threshold. Averageswere based on either ®ve trials (1.25 and 1.56T) ortwo to three trials (36T condition). The axis of theelectrode was aligned with the axis of the vertebralcolumn. The cathode was located caudally. Thismethod of spinal cord stimulation most likelyexcites motor axons directly at the level of the axonhillock (Mills and Murray, 1986; Kimura, 1989).

Measurement and analysis of MEPsSingle trial MEP records were full-wave recti®edand averaged (Figure 1). Recti®cation avoidspotential cancellation of components in raw EMGsignals that could affect magnitude measurements.MEP onset latency was computer measured fromaveraged records. Cord MEP onset latency providesa measure of peripheral conduction time whilecortical MEP latency provides a measure totalconduction time. As illustrated in Figure 5, we alsoobtained a measure of central conduction time(CCT) by subtracting spinal cord MEP latency fromcortical MEP latency. Cortical MEP magnitude wasquanti®ed by expressing the peak and area as apercentage of the maximal cord MEP obtained at36threshold. Cortical MEPs were generally about15% the magnitude of maximal cord MEPs.

Statistical analysisQuantitation of baseline variability in spinal cordand cortical MEPs was based on data collected fromfour monkeys that served as controls over a periodof 6 months. The signi®cance of post-inoculationchanges in MEP latency, magnitude and thresholdwas interpreted in relation to the standard deviationof data from control monkeys. Post-inoculationMEP latencies, magnitudes and thresholds thatexceeded two standard deviations of control values

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were judged to be signi®cant. The statisticalanalyses were designed to provide descriptions ofthe relationship between SIV infection and MEPoutcomes and are considered exploratory ratherthan inferential. Both single subject and populationanalyses were performed. MEP latencies for indivi-dual animals were examined graphically usingprocedures analogous to Shewert control chartsoften used in industrial process control (Box et al,1978). To generate estimates of within monkeyvariability needed to construct the control charts,a variance components analysis was applied to thepre-inoculation observations from all nine monkeysto partition MEP latency variability into between-animal and within-animal components. Controlcharts for each animal were then constructed usingthe mean pre-inoculation latency for that animaland the average within-monkey standard deviationobtained from the variance components analysis.Plots of the post-inoculation latencies were thencompared to the expected range (mean+2 standarddeviations) to assess whether SIV infection wasindicative of MEP pathophysiology. Separate ana-lyses were conducted for each MEP type (sourceand muscle) for the 1.25 threshold condition.

Histology and neuropathologyAt necropsy, monkeys were given an initial dose ofketamine (10 mg/kg) followed by a near lethal, I.V.dose of pentobarbital. All animals (except AQ47and AQ43) were exsanguinated through the des-cending aorta and perfused transcardially with twoliters of normal saline followed by two liters of 10%neutral buffered formalin. AQ47 and AQ43 were notperfused with formalin so that fresh tissue samplescould be taken for molecular analysis. Following®xation either by perfusion, or in the case of AQ47and AQ43 immersion, the brain, spinal cord andsome peripheral nerves (median, tibial, sciatic andfemoral) were removed and processed for histo-pathological analysis.

From an earlier constructed dissection map of thetypical macaque brain, the right hemispheres weredissected at 10 mm intervals in the coronal planeproviding access to the following regions of themotor pathway: the motor and pre-motor cortices,

the basal ganglia (including the caudate, putamenand globus pallidus), thalamus and the deep whitematter (including the internal and external cap-sules, comissural ®bers and the periventricularwhite matter). The brainstem was dissected trans-versely at 5 mm intervals and samples included theupper and lower mid-brain at the levels of thesuperior and inferior colliculi, the mid-pons and theupper and lower medulla (including the pyramidsand lower cranial nerves). The spinal cord wassectioned transversely at 10 mm intervals throughthe cervical (C2 and C7/C8), thoracic (T2, T6 andT10) and lumbar (L2 and L5) segments. Afterembedding the tissue in paraf®n, 5 mm serialsections were stained with hematoxylin and eosin(H&E), Luxol Fast Blue and Sevier Munger stains toassess suspected neuropathology. The severity ofmotor system pathology was scored in a semi-quantitative fashion based on the number, sizeand type of lesions per microscopic ®eld.

Two centimeter long segments of the median,sciatic, posterior tibial and femoral (AQ43 only)nerves were removed, embedded in paraf®n andsectioned ®rst transversely and then longitudinallyat 5 mm. Three to six transverse sections and threeserial longitudinal sections were examined for eachnerve. The sections were mounted on gelatin coatedslides, stained with H and E, and examined forevidence of axonal loss and/or demyelination usinglight microscopy. A complete report of the neuro-pathology is available elsewhere (Ragahavan et al,1999).

Acknowledgements

The authors thank Randall Lininger and JillBrandom-Harris for their expert technical contri-butions. In addition, we thank Jim Rengel and TedGleason for assistance with electronics, Drs DavidPinson and Istavan Adany, for their assistancewith the necropsies and tissue cataloging and DrRichard Dubinsky for help in evaluating neurolo-gical symptoms. This research was supported byNIH grants NS32203 and HD02528.

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