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Journal of NeuroVirology, 7: 265271, 2001c 2001 Taylor & Francis ISSN 13550284/01 $12.00+.00

Age-dependent poliomyelitis in mice is associatedwith respiratory failure and viral replication in thecentral nervous system and lung

EH Schlenker,1 QA Jones,1 RRR Rowland,2 M Steffen-Bien,2 and WA Cafruny1

1Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, South Dakota, USAand2Department of Biology and Microbiology, South Dakota State University, Brookings, South Dakota, USA

Age-dependent poliomyelitis (ADPM) is a virally induced neuroparalytic dis-ease of mice and a model for amyotrophic lateral sclerosis (ALS). ADPM istriggered in genetically susceptible mice by immunosuppression and infectionwith lactate dehydrogenase-elevating virus (LDV). Both ADPM and ALS arecharacterized by progressive degeneration of anterior horn motor neurons,

and death in ALS is usually associated with respiratory failure. To assess res-piratory function in ADPM, we investigated ventilation in conscious controland LDV-infectedC58/J mice breathing air and then 6.5% CO2 in O2. Threedaysafter LDV infection, ventilation in response to CO2 was half of that comparedto the uninfected state, but become normalized by 10 days. Administration ofcyclophosphamide alone (200 mg/kg, ip), an immunosuppressant, had no ef-fect on ventilation. Induction of ADPM by concomitant administration of LDVto cyclophosphamide-treated mice resulted in altered gait, hindlimb paralysis,wasting, decreased metabolism, and decreased body temperature by 4C rela-tive to controls. Compared to baseline values, mice with ADPM had decreasedtidal volume and ventilation while breathing air, and while exposed to the CO2challenge they were unable to increase tidal volume, frequency of breathing,or ventilation. Using in situ hybridization, LDV replication was noted withinthe spinal cord, brain, and lung, but not in the diaphragm. Thus, respiratory

failure is a contributory mechanism leading to death in ADPM and is associ-ated with LDV replication in the CNS and lung. This animal model may beuseful to investigate physiological and molecular mechanisms associated withthe development of respiratory failure in neurodegenerative diseases.Journalof NeuroVirology(2001) 7, 265271.

Keywords: respiration; ALS; ADPM; LDV; metabolism

Introduction

Age-dependent poliomyelitis (ADPM) is a motorneuron disease of mice (reviewed in Contag et al,1989) that involves the interaction of two viruses.In many respects, ADPM is similar to amyotrophiclateral sclerosis (ALS) because both diseases are age-related, have genetic and environmental risk fac-tors, and share anterior horn neurons as the pri-mary pathological targets (Sillevis Smitt and de

Jong, 1989; Bromberg, 1999). The etiology of ALSis not known, although recent evidence suggests thepresence of enterovirus (probably echovirus 7) RNA

Address correspondence to E. H. Schlenker, Division of

Basic Biomedical Sciences, University of South Dakota School ofMedicine, Vermillion, SD 57069, USA.Received 28 June 2000; revised 12 October 2000; 17 January

2001.

in the spinal cords of ALS patients, raising the pos-sibility that ALS is a viral disease (Berger et al, 2000;

Karpati and Dalakas, 2000). Respiratory failure is thecommon cause of death in ALS (Ferguson et al, 1996;Bromberg, 1999; Arnulf et al, 2000), but the causeof death in ADPM is not known, despite previousspeculation that respiratory paralysis may play a role(Sillevis Smitt and de Jong, 1989; Anderson et al,1995a).

ADPM has a complex etiology. It results fromlactate dehydrogenase-elevating virus (LDV) infec-tion of genetically susceptible strains of mice (e.g.,C58 and AKR), which express two copies of theFv-1n=n allele that regulates permissiveness to en-dogenous retrovirus replication (Martinez etal, 1980;

Murphy et al, 1980; Nawrocki et al, 1980; Contagetal, 1989; Plagemann and Moennig, 1992; Andersonet al, 1995a; Anderson et al, 1995b; Plagemann,

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Virus-induced poliomyelitis and respiratory failure

266 EH Schlenker et al

1996). As these mice age, they develop susceptibility to ADPM induction by LDV infection, dueto immunosuppression associated with aging. Treat-ment with cyclophosphamide or X-irradiation caninduce immunosuppression of younger mice and en-hance susceptibility to ADPM (Contag et al, 1989;Plagemann and Moennig, 1992; Plagemann, 1996).

Infection of susceptible mice with neuroparalyticstrains of LDV induces a systemic infection and entryof LDV into the CNS. This is followed by a subclin-ical phase of about 10 days, during which LDV isnot present at pathologic levels in the CNS. At about1012 days postinfection (p.i.) with LDV, LDV repli-cation emerges in the CNS. There is a brief pro-drome during which mice appear sluggish and beginto lose weight (wasting). Between about 1218 days,p.i. mice develop frank paralysis (typically hindlimb)and die within a few days to weeks.

Although LDV appears to have the primary role

in neuropathology, ADPM is clearly a dual-virus dis-ease (Contag et al, 1989; Plagemann and Moennig,1992; Plagemann, 1996) that requires some degree ofimmunosuppression. Thus, ADPM provides an at-tractive small animal model for degenerative neu-rological diseases that involve aging and potentialvirus etiologies. The present study documents thedevelopment of respiratory failure in ADPM, whichcorrelated with LDV replication in lung cells andcentral nervous system neurons. To determine if de-pression of metabolic rates coincided with the ven-tilatory effects of ADPM, both CO2 production andO2 consumption were measured in the mice. Con-

trol studies were conducted to determine if LDVinfection or cyclophosphamide alone had similarphysiological effects as the combination of the twotreatments.

Methods

Eight-month-old female C58/J mice (The JacksonLaboratory) were used in these experiments. Micewere housed 34 per cage. Food (Purina MouseChow) and water were available ad libitum. Light-

ing consisted of 12 h on and 12 h off. The Univer-sity of South Dakota Animal Care and Use Committeeapproved all procedures.

Ventilatory and metabolic measurementsTo evaluate ventilation, mice were placed in a Plexi-glas cylindrical chamber 15 cm long and 6.35 cm indiameter. The chamber had three ports on one sidefor an air inlet, a Statham low-pressure transducer,and a Cole-Palmer thermometer to measure chambertemperature. On the opposite side, ports allowed airto exit the chamber to a Gilmont rotameter and an-other line that was either connected to gas analyz-

ers (OM-14 Beckman oxygen analyzer or a vacuumedCO2 analyzer) or to a leak used to stabilize ventilatorymeasurements.

The Statham transducer was connected to a Grassmodel 7 polygraph. Outputs from the polygraph wereintroduced to an MP100 Biopac data analysis sys-tem. The parameters evaluated included breathingfrequency (f ) in breaths/min, tidal volume (Vt) inml/breath, and minute ventilation (VE), the productof Vt and f in ml/min.

Oxygen consumption and CO2 production weremeasuredusing the ow-through technique. For bothevaluations, the differences between the fractionalconcentrations of gases entering and exiting thechamber were measured and multiplied by the owrate through the chamber. These methods have beenreported previously (Schlenker, 1984).

ProtocolsThe animals were weighed prior to being placedinto the plethysmographic chamber. First, the mousewas acclimated to the chamber and ventilatory and

metabolic measurements were conducted. Then, themouse was exposed for 30 s to 6.5% CO2 in O2 tomeasure the response to hypercapnia. Subsequently,the mouse was exposed to air again. After the mousewas removed from the chamber its rectal tempera-ture was measured using a Sensortek thermometer(Clifton, NJ).

Treatment groups and virus infectionsPrior to any treatments, baseline ventilatory andmetabolic measurements were performed on allanimals. Three treatment groups were studied. Avirus-only group (10 C58/J mice total) received an

intraperitoneal injection of LDV v-9 (106 ID50). LDV-v9 is a neuroparalytic (Group B) quasispecies iso-lated from the spinal cord of a paralyzed C58 mouseinfected with the cloned quasispecies LDV-v (Chenet al, 1999). LDV-v9 stocks were generated from theplasma of CF1 mice (Harlan SpragueDawley) 24 hp.i. with LDV-v9 and titrated by limiting dilution as-say (Plagemann and Moennig, 1992). In the secondgroup, 10 C58/J mice received an intraperitoneal in-jection of 200 mg/kg cyclophosphamide on day 1 anda vehicle injection on day 2. A third group of sixC58/J mice received cyclophosphamide on day 1 and

LDV-v9 on day 2. On day 7, cyclophosphamidewas once again administered to groups 2 and 3.Physiological measurements were conducted priorto cyclophosphamide administration and on days3, 5, 7, and 12 after virus treatment until micein group 3 developed severe disease on 16 to20 days after infection. Subsequently, LDV andcyclophosphamide-treatedmice, and animals treatedwith cyclophosphamide alone, were sacriced.Brains, lungs, and diaphragms were quickly re-moved, xed in 10% buffered formalin, and preparedfor LDV RNA localization.

Localization of LDV RNA in tissuesIn situ hybridization was used to determine ifLDV replication was present in the brain, spinal

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EH Schlenker et al 267

Table 1 Effect of cyclophosphamide and LDV infection on O2 consumption (VO2) and CO2 production (VCO2)

Baseline Day 16

Treatment groups VO2 VCO2 VO2 VCO2

Cyclophosphamide alone 1.91 0.39 1.38 0.10 1.63 0.41 1.52 0.19Cyclophosphamide alone and LDV 2.00 0.46 1.65 0.45 1.05 0.22 0.86 0.24

O2 consumption (VO2) and CO2 production (VCO2) are in units of ml/min. There were six animals per group. The asterisks denotes thatthe day 16 VO2 is signicantly less than baseline value in the same group of animals and that the day 16 VCO2 is signicantly less thanbaseline value in the same group of animals. Values are means SD.

cord, lung, or diaphragm. Tissues were xed in for-malin, embedded in parafn, sectioned at 6 um,and then probed with a 35S-labeled cDNA probe(#4-55) that hybridizes to LDV genomic RNA and allsevensubgenomic RNAs (Kuo etal, 1992). Autoradio-graphically processed sections were stained withhematoxylin and eosin and examined under a lightmicroscope for localization of LDV RNA and es-

timation of tissue virus burden. Uninfected tissuesections were prepared as internal experimentalcontrols.

Quantication of tissue LDV virionsPlasma, lung, diaphragm, brain stem, and cervicalspinal cord samples were obtained from mice sac-riced during paralysis at 16 days p.i. with LDV-v9.Tissue samples (except for plasma) were washed inPBS, weighed, and minced with a hollow glass rod,which was used to completely grind and disrupt thetissues in the bottom of a 12 75 mm tube contain-

ing cold PBS. These tissue extracts were then clar-ied by centrifugation at 1000 g for 5 min. Thequantitative method of choice for LDV virions is ahighly sensitive end-point dilution assay, in which10-fold dilutions of samples are injected into indica-tor mice, which display elevated plasma LDH afterLDV infection (Rowson and Mahy, 1975; Plagemannand Moenning, 1992). Therefore, LDV titers were de-termined in plasma and claried tissue extracts byend-point dilution analysis, using two mice for eachlog10 dilution.

Data analysisTo determine if each treatment had an effect of

body weight, body temperature, CO2 production andO2 consumption or ventilatory parameters, a pairedt-test was used to compare baseline and nal values.Signicance was accepted at P< 0:05. Values are ex-pressed as means SD.

Results

Body weights, body temperatures, and metabolismTreatment of mice with LDV alone or with cyclophos-

phamide alone had no signicant effects on metabolicparameters, body temperature, or body weight. Incontrast, animals treated with cyclophosphamide

and LDV exhibited a marked decrease in body weightfrom 23.3 0.8 to 18.3 0.5g (mean SD), P< 0:01,which occurred between about 1620 days post in-fection (p.i.). Moreover, body temperature droppedfrom a baseline value of 37.7 0.17 to 33.1 2.9 C( P< 0:01) within the same time frame. The effectsof treatment with cyclophosphamide and LDV, or cy-clophosphamide alone, on CO2 production and O2consumption are presented in Table 1. Baseline val-ues were comparable between the two groups. Byday 16 after treatment started (the paralytic phaseof ADPM), CO2 production and O2 consumptionin cyclophosphamide- and LDV-treated mice weremarkedly lower than their baseline values and alsolower than those of cyclophosphamide-treated mice.The mice progressing to ADPM exhibited a wastedappearance with a slowing of gait and developedhind-limb paralysis.

Control of breathing

Control of breathing in mice exposed to air andto hypercapnia was not affected by one or twodoses of cyclophosphamide (data not shown). Theventilation in response to LDV alone revealed twotypes of responses (Figure 1). Half of the micegiven LDV showed a transient decrease of ventila-tion in response to hypercapnia by day 3. This de-crease was due to a decreased Vt and f. Mice in-fected with LDV alone did not develop paralysis.The decreased ventilatory responses to hypercap-nia in these mice returned to baseline values by10 days p.i.

Mice given cyclophosphamideplus LDV decreasedtheir Vt, but not f, while breathing air by day 15 afterinfection (Table 2). Ventilatory responses to hyper-capnia were similar to those of the mice while breath-ing air or blunted (Figure 2). The inability to respondto hypercapnia was due to a marked reduction of Vtand f. The time period for this blunted response tohypercapnia ranged from 15 to 21 days after infec-tion. Mice either died or were sacriced shortly afterdecreasedventilation was recorded, and all mice dis-played a wasted appearance and a halting gait.

Localization of LDV RNA and tissue virions

In situ hybridization demonstrated LDV RNA inneurons within the brain and spinal cord, and inthe lung coincident with the onset of LDV-induced

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Figure 1 Effects of lactate dehydrogenase-elevating virus (LDV)on ventilation in mice exposed to air (A) and CO2 (C) at baselineand 3 days after infection. The left panel denotes the ve animalsthat exhibited a signicant (P< 0:01) depression of ventilationon

day 3 on response to CO2 (as denoted by the asterisk). These arecalled the responders. The right panel denotes the ve animals(nonresponders) that did not exhibit a depression of ventilation3 days after being infected with LDV.

paralysis (representative data from ve mice withADPM are shown in Figure 3). The occasionally elon-gated pattern of LDV RNA in spinal cord sections (ar-rows, Figure 3C and F) is suggestive of nerve axonlocalization. In contrast, LDV RNA was not detectedin the diaphragms of any of the mice studied butwas detected within a lymph node located in the di-

aphragm (arrows, Figure 3B and E). The lung andlymph nodes within the lung contained LDV RNA.

Figure 2 Effects of lactate dehydrogenase-elevatingvirus (LDV) and cyclophosphamide treatment on ventilation in mice exposed to airand CO2 at baseline and 16 days after infection when paralysis or altered gait was o bserved. The asterisk denotes a signicant (P< 0:01)ventilatory response to hypercapnia. There were six animals in this group.

Table 2 Effect of c yclophosphamide and LDV infection on tidalvolume (Vt) and frequency of breathing (f)

Baseline Paralysis

Vt (air) 0.25 0.05 0.16 0.03Vt (CO2) 0.39 0.08 0.19 0.03f (air) 223 28 227 53

f (CO2 ) 318 44 255 49

Values are means SD. The asterisks indicate that the values aresignicantly different (P< 0:05) from the corresponding baselinevalue. Paralysis decreases Vt (ml/b) in air and both Vt and F(b/min) in response of mice to hypercapnia (CO2 ). There were sixanimals per treatment group.

The proportion of lung cells expressing LDV RNAranged from about 58/10000. Since these cells areproductively infected, theyrepresent a theoretical tis-sue virus burden of 58 104 virus particles/10000lung cells (Plagemann and Moennig, 1992). As re-ported previously (Anderson et al, 1995a), brain andspinal cord LDV RNA was detected in multiple foci(37 per hemisphere or spinal cordcross-section), butdue to the inherent lack of histological resolution,no estimation of the proportion cells productivelyinfected was possible for these neural sites of virusreplication. Cyclophosphamide-treated mice thatwere used as controls did not exhibit LDV RNA inany of the tissues examined. Tissue virions were alsoquantitated in two mice during LDV-induced paral-ysis at 16 days p.i. with LDV (Table 3). By this ap-proach, large viral burdens were seen in all tissues

examined, potentially reecting local replication orvirus from the circulation.

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Figure 3 Detection of lactate dehydrogenase-elevatingvirus (LDV) RNA in lung, diaphragm, and spinal cord by in situ hybridization.Tissues of paralyzed C58 mice 16 days postinfection with LDV. LDV RNA was consistently found in the lung (A and D). No LDV RNAwas detected in the diaphragm (B and E), although LDV RNA-positive lymph nodes were present in both diaphragm (E) and lung (D).Detection of LDV RNA in spinal cord (C and F) suggested a pattern consistent with axonal LDV replication.

Discussion

We report here the rst description of respiratoryfailure in ADPM, extending the similarities betweenmurine ADPM and human ALS (Sillevis Smitt and

de Jong, 1989; Ferguson et al, 1996). Mice treatedwith a combination of cyclophosphamide and LDVdeveloped wasting, altered gait, and hind-limb paral-ysis (ADPM as previously described). Accompany-ing these manifestations were decreasedbody weight,

body temperature, O2 consumption and CO2 produc-tion, and ventilation in air and in response to hyper-capnia. In contrast, treatment of mice with LDV or cy-clophosphamide alone did not elicit these changes.Moreover, during paralysis, LDV RNA was located

Table 3 Tissue virus levels during ADPM

Tissue Virion Concentration1

Tissue Mouse A Mouse B

Plasma 107:0 ID50/ml 106:0 ID50/ml

Lung 105:9 ID50/g 105:4 ID50/g

Diaphragm 106:4 ID50/g 106:4 ID50/g

Spinal cord 106:2 ID50/g 106:6 ID50/g

Brain stem 105:3 ID50/g 106:8 ID50/g

Two 78-month-old female C58/J mice were treated with cy-clophosphamide 200 mg/kg (days 1 and 7) and LDV infectedon day 0. Both mice were sacriced on day 16 p.i., at whichtime both exhibited limb paralysis, wasting, and respiratorydysfunction. After washing of individual solid tissue samples,

virus was extracted by extensive mincing in cold PBS.1 Tissue LDV titers were determined by limiting dilution titration,and are reported as ID50/ml (plasma) or ID50/g of wet weight (allother tissues).

in the spinal cords, brains, and lungs but not withinthe diaphragms of these animals, demonstrating pro-ductive LDV infection at all sites but the diaphragm.Consistent with previous studies (Rowson and Mahy,1975), LDV virions were found in all tissue sites in-

cluding diaphragm, but the absence of LDV replica-tion in diaphragm illustrates the limitations of viriondetection, which may reect virus delivered from thecirculation as well as locally produced virus. Whilethese data raise the possibility of synergy betweensite-specic productive infection and passive accu-mulation of virions in contributing to pathology, theyprimarily emphasize the signicance ofin situ detec-tion of LDV replication, which is cytocidal and there-fore directly pathologic.

The lungs as a reservoir for LDV replication

The emergence of LDV replication in the CNS justprior to onset of paralysis has been described previ-ously as a likely neuropathogenic mechanism (Chenet al, 1999). This is signicant since after the acutephase of infection, virus replication in most mousetissues is almost undetectable, due to exhaustionof LDV-permissive cells about 24 h after infection(Plagemann and Moennig, 1992). Our data demon-strate the lung is an additional target organ for per-sistent LDV replication in ADPM. Although in situhybridization does not yield a precise quantitative re-sult, the proportion of LDV RNA-producing cells wasestimated to approach 1/1000, representing a signif-

icant virus reservoir. The physiological signicanceof LDV replication in the lung is not known, but itmay affect lung mechanics (and thereby the work of

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breathing) and gas exchange characteristics (Arnulfet al, 2000). Future studies will investigate these pos-sibilities throughout the course of infection.

Effects of virus infection on control of breathingThe decrement in ventilation of LDV- and

cyclophosphamide-treated mice may be due toseveral factors. These include decreased metabolicdemand, decreased body temperature, loss of res-piratory muscle mass or functional capabilities,and/or abnormalities in CNS areas associated withregulation of breathing, metabolism, and regulationof body temperature (Mortola and Gautier, 1995).The underlying mechanisms by which local virusreplication in the lung or within CNS contributeto respiratory failure in ADPM are unclear, but ourdata clearly show that the lung is an importanttarget organ for ADPM-associated pathology, andthat respiratory failure may represent the cause ofdeath in mice with ADPM.

ADPM has interesting characteristics as a modelfor human degenerative diseases of viral or unknownorigin: it is a dual virus disease (i.e., requires bothLDV and retrovirus replication as necessary cofac-tors), andis dependent upon somedegree of immuno-suppression in the host (provided by natural agingor induced immunosuppression). Moreover, ADPMprovides a model to study how viral-induced fac-tors, nerve degeneration, and potential cytokine al-terations, may contribute to the development of res-piratory failure. LDV infection results in disturbance

of various cytokines (Monteyne et al, 1993), as wellas immunological alterations that may impact cy-tokine levels (Plagemann and Moennig, 1992). Cy-tokines such as tumor necrosis factor alpha and in-terferons regulate LDV replication (Plagemann andMoennig, 1992; Cafruny et al, 1997), and can also in-teract with hormones including leptin, growth hor-mone, and thyroid hormones that affect food in-take, body weight, and metabolic rate (Boelen Aet al, 1995; Wassen et al, 1996; Johnson et al, 1997;Sarrafet al, 1997; Raina and Jeejeebhoy, 1998; Finckand Johnson, 2000). Within the brain, cytokines in-uence the function of neurotransmitters and re-ceptors such as opioids and gamma aminobutyricacid (GABA) that depress respiration and modu-late metabolism (McCrimmon et al, 1995; Rothwelland Hopkins, 1995; Schlenker and Inamdar, 1995;Kalra et al, 1999; Vitkovic et al, 2000). Interleukinsalso affect neuroendocrine function causing the

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