clinical efficacy of buprenorphine to minimize distress in mrl/lpr mice

logy 567 (2007) 67–76www.elsevier.com/locate/ejphar

European Journal of Pharmaco

Clinical efficacy of buprenorphine to minimize distress in MRL/lpr mice☆

Julie Swenson a,1, Selen Olgun b, Ali Radjavi a, Taranjit Kaur a, Christopher M. Reilly a,b,⁎

a Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine,Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, United Statesb Edward Via College of Osteopathic Medicine, Blacksburg, VA 24060, United States

Received 28 September 2006; received in revised form 4 March 2007; accepted 6 March 2007Available online 6 April 2007

Abstract

MRL/MpJ-Faslpr (MRL/lpr) mice are an accepted animal model to study human systemic lupus erythematosus. We tested if a commonly usedanalgesic (buprenorphine hydrochloride) would reduce pain and distress in these mice without impacting the progression of autoimmune disease.Female MRL/lpr mice were randomly separated into four groups. Experimental groups received cyclophosphamide (25 mg/kg i.p. weekly),buprenorphine (0.09 mg/kg/mouse/day via drinking water), or cyclophosphamide+buprenorphine from 11 to 21 weeks of age. Controls receivedno treatments. Mice were monitored daily by a licensed veterinarian (blinded observer) and assigned a score weekly on parameters associated withpain and distress as well as progression of disease. Proteinuria was measured weekly, and serum anti-dsDNA antibody levels were determined at11, 15, and 18 weeks of age. At 21 weeks of age, the animals were euthanized and the kidneys and spleens were removed for evaluation.Regardless of the parameter observed, buprenorphine did not significantly decrease distress when compared to the controls. Buprenorphine did notalter the progression of autoimmune disease, based on characteristics of splenic architecture and splenocyte cell profiles, development oflymphadenopathy, or kidney histology as compared to controls. This study indicates that buprenorphine at this dose and route of administrationwas ineffective in reducing distress associated with disease progression in the MRL/lpr strain. More studies are needed to determine if, at adifferent dose or route, buprenorphine would be useful as adjunctive therapy in reducing distress in MRL/lpr mice.© 2007 Elsevier B.V. All rights reserved.

Keywords: Buprenorphine; MRL/lpr; Distress; Systemic lupus erythematosus; Pruritus

1. Introduction

MRL/MpJ-Faslpr (MRL/lpr) mice spontaneously develop anautoimmune syndrome similar to human lupus erythematosusincluding auto-antibody production and immune complexglomerulonephritis and are commonly used as a murine modelto study lupus (Reilly and Gilkeson, 2002). MRL/lpr mice beginshowing overt signs of disease by 3 months of age. Along withauto-antibody production and immune complex glomerulone-

☆ This material has not been published and/or is not under active considerationby another journal. The research conducted was in accordance with the Guidefor the Care and Use of Laboratory Animals as adopted and promulgated by theUnited States National Institutes of Health.⁎ Corresponding author. Edward Via College of Osteopathic Medicine, 2265

Kraft Drive, Blacksburg, VA 24060, United States. Tel.: +1 540 231 5345; fax:+1 540 231 5252.

E-mail address: [email protected] (C.M. Reilly).1 Current Address: Zoological Medicine Service, Veterinary Medical

Teaching Hospital, College of Veterinary Medicine, Kansas State University,Manhattan, Kansas 66506, United States.

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.03.048

phritis, MRL/lpr mice develop polyarthritis, severe lymphade-nopathy, hypergammaglobulinemia, proteinuria, skin lesionswith alopecia, scab formation, and erythematous lesions of theear pinna that often become necrotic as the mice age (Andrewset al., 1978; Molano et al., 2003; Okamoto et al., 2004; Reillyet al., 2000, 2002; Sekine et al., 2001;Watanabe-Fukunaga et al.,1992). MRL/lpr mice also show severe excoriations of the skindue to self-trauma associated with excessive pruritus, which isthought to be similar to the pruritus seen in humans withautoimmune diseases, such as systemic lupus erythematosus andprimary biliary cirrhosis (Kapadia and Haroon, 1996; Umeuchiet al., 2005). These lesions interfere with species specificbehaviors, leading to increasing distress in the mice.

Few reports exist concerning methods to minimize pain anddistress in MRL/lpr mice that do not interfere with the study ofdisease progression for research purposes. Many compoundsused in laboratory animals to minimize pain and distress havesignificant disadvantages for treating large colonies of animals,including frequent dosing schedules and/or the need to use

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http://dx.doi.org/10.1016/j.ejphar.2007.03.048

68 J. Swenson et al. / European Journal of Pharmacology 567 (2007) 67–76

injectable routes of administration (Christoph et al., 2005;Cooper et al., 1997; Gades et al., 2000; Gross et al., 2003;Roughan and Flecknell, 2004). These problems contributesignificantly to the difficulties in managing pain and distress inmurine models of autoimmune disease.

Buprenorphine hydrochloride is a potent opioid analgesic withpartial agonistic properties at μ-opioid receptors (Ide et al., 2004;Pick et al., 1997). Buprenorphine has been shown to have anantinociceptive potency 25 to 40 times higher than morphine(Cowan, 2003). Benefits of buprenorphine include a high analgesicpotency with a long duration of action (Kogel et al., 2005) andformulation as a water soluble compound that can be administeredorally in the drinking water (Cooper et al., 1997; Lindsay et al.,2005). The use of buprenorphine as an analgesic has shownefficacy in rodent models of acute and chronic pain, includingvisceral, somatic, and inflammatory pain (Christoph et al., 2005;Roughan and Flecknell, 2004). Buprenorphine has also beenshown to attenuate self-injurious behaviors in mouse models ofpsychostimulant addiction and, although there have been nocontrolled studies of its use in humans for treatment of systemiclupus erythematosus, it is thought to be an appropriate drug ofchoice in humans suffering from other illnesses that stimulate self-trauma (Mori et al., 2006).A recent study inmicewith experimentalToxoplasma gondii infection has indicated that clinical signs of painand distress are reduced when buprenorphine is administered viathe drinking water without interfering with mortality rates ascompared to untreated controls (Lindsay et al., 2005).

The purpose of this study was to determine if buprenorphinehydrochloride could be used to alleviate pain and distress inMRL/lpr mice without negatively impacting common researchparameters, including immune system function, or progressionof disease.

2. Materials and methods

2.1. Animals

Eight week old MRL/lpr female mice were purchased fromJackson Laboratories. The mice were housed in specificpathogen free conditions at the Center for Molecular Medicineand Infectious Diseases animal facility at Virginia Polytechnicand State University. They were housed in a 12 h light:darkcycle and provided food and water ad libitum. All animalprotocols were approved by the Institutional Animal Care andUse Committee at the Virginia Polytechnic and State University.

2.2. Reagents

DMEM and RPMI were obtained from Gibco (Gaithersburg,MD). The protein assay kit was purchased from Bio Rad(Hercules, CA). All other reagents were purchased from Sigma(St. Louis, MO).

2.3. Urine protein analysis

Urine protein determinations were made using Chemstrip® 2GP reagent strips from Roche (Indianapolis, Indiana).

2.4. Study groups

Forty mice were randomly separated into 8 cages, with 5mice per cage, and 2 cages in each study group for a total of10 mice per study group. At 11 weeks of age, the micereceived cyclophosphamide, buprenorphine, or cyclophos-phamide+buprenorphine. The control group received notreatments. Buprenorphine hydrochloride (Abbott Laborato-ries, North Chicago, Illinois) was administered via thedrinking water to deliver 0.09 mg/kg per mouse per day(Cooper et al., 1997; Lindsay et al., 2005). Clean bottles withfreshly prepared buprenorphine-treated water were providedevery 3 days as a method to help ensure potency of the drug.Green-tinted water bottles were used to prevent decay ofbuprenorphine, which is a light sensitive compound. Cyclo-phosphamide (25 mg/kg BW) was given weekly to thecyclophosphamide and cyclophosphamide+buprenorphinegroups via intraperitoneal injection in 40 μl saline.

2.5. Animal observations

All study animals were observed daily for evidence ofextreme pain and distress by the veterinarian, who was blindedto the treatment regime. Extreme pain and/or distress wasdefined as pain and/or distress that exceeded what is considerednormal for disease progression in MRL/lpr mice and wasindicated by any one of the following: 1 — open skin lesionsthat would not stop bleeding, 2 — evidence of significantrespiratory distress such as abdominal breathing and/or cyanosisof the mucous membranes, or 3 — any animal that was non-responsive to stimulation. During the course of the study, twoanimals were removed due to extreme pain and distress. Thefirst animal was in severe respiratory distress and waseuthanized 1 week prior to the termination of the study. Thesecond animal was euthanized due to uncontrollable bleedingsecondary to self-mutilation 1 day prior to termination of thestudy.

Once weekly, each animal was given a complete physicalexamination. The physical examination included observationwithin its cage and palpation once removed from its cage. Theseobservations were scored using a numerical scoring system thatwas developed from the current animal care policy for MRL/lprmice housed at Virginia Polytechnic Institute and StateUniversity at the time of this study. The scoring criteria arelisted below.

2.5.1. Body condition scoringBody condition scoring was done using a 5 point scale as

described previously (Ullman-Cullere and Foltz, 1999). A bodycondition score (BCS) of 3/5 was considered within normallimits for mice on this study. A BCS of 1/5 represented anemaciated animal, 2/5 represented an underweight animal, 4/5indicated an overweight animal, and 5/5 indicated an obeseanimal. As an indicator of pain or distress, only a bodycondition score of 2/5 or less was considered significant. Ifconsidered significant, the BCS was added to the combineddistress score as follows: 1 = BCS of 2/5, 2 = BCS 1/5.

Fig. 1. Characterization of skin lesions was based on a numerical scoring systemfrom 0 to 3 (no lesions to severe lesions, respectively). The animal shown here(A) was scored as a 2 for moderate lesions (defined as scabbing over b5% of theskin surface). (B) Tabulation of skin lesion scores. No significant differenceswere seen between the four treatment groups based on skin lesions (Pb0.05).Each group in (B) represents the average of at least 8 animals.

Fig. 2. Tabulation of scores for other common indicators of distress scores andlymphadenopathy. (A) Other common indicators of stress included abnormalrespiratory rate and effort, presence of a ruffled haircoat, presence of huddlingbehavior, and noticeable pruritus. Each of these parameters was scored on thenumerical scoring system listed below. Respiratory rate and effort: 0 = normalrespiratory rate and effort, 1 = increased respiratory rate, but normal effort, 2 =increased respiratory rate and effort, 3 = increased respiratory rate and effortwith an abdominal component to the breathing. Ruffled haircoat, huddlingbehavior, and pruritus: 0 = absence of the indicator, and 1 = presence of theindicator. These scores were combined together to form a score titled “otherindicators of stress”. There were no significant differences noted between thefour treatment groups based on other indicators of distress (Pb0.05). (B)Lymphadenopathy was scored on a numerical scoring system from 0 to 3 (noneto severe enlargement, respectively). There were no significant differences notedbetween the four treatment groups based on the development of lymphadenop-athy (Pb0.05). Each group in (A) and (B) represents the average of at least8 animals.

69J. Swenson et al. / European Journal of Pharmacology 567 (2007) 67–76

2.5.2. Mentation scoringMentation was assessed using the following parameters:

BAR = Bright, alert, and responsive (BAR indicated normalactivity level and mentation for a mouse); QAR = Quiet, alert,and responsive (QAR indicated an animal that was less activethan normal while under observation in the cage, but becamemore active upon stimulation); DR = Depressed, but responsive(DR indicated an animal who would respond to stimulation, butremained huddled on the cage bottom); and NR = Non-responsive (NR indicated a conscious animal that would notrespond even with significant stimulation). Each animal wasgiven a score to represent their individual level of mentation. Thescoring system used for mentation was as follows: 0 = BAR, 1 =QAR, 2 = DR, and 3 = NR.

2.5.3. Characterization of skin lesionsMice were individually examined for evidence of skin

disease and self-trauma. The lesions were scored based on thefollowing: 0 = no lesions noted, 1 = mild lesions (alopecia withsmall amount of scabbing present), 2 = moderate lesions

(scabbing over b5% of the skin surface), and 3 = severe lesions(scabbing over N5% of the skin surface).

2.5.4. Other common indicators of stressOther common indicators of stress in rodents were noted

during the weekly physical exam. These included abnormalrespiratory rate and effort, presence of a ruffled haircoat,presence of huddling behavior, and noticeable pruritus. Each ofthese parameters was evaluated prior to any manipulation andscored using the following criteria. Respiratory rate and effortwas scored as follows: 0 = normal respiratory rate and effort, 1 =increased respiratory rate, but normal effort, 2 = increasedrespiratory rate and effort, 3 = increased respiratory rate andeffort with an abdominal component to the breathing. For theruffled haircoat, huddling behavior, and pruritus parameters, themice were scored as follows: 0 = absence of the indicator, and 1 =presence of the indicator. These scores were combined togetherto form a score titled “other indicators of stress”.

Fig. 3. Tabulation of scores for mentation and combined distress scores. (A)Mentation scores were significantly higher in the buprenorphine and the controlgroups when compared to the cyclophosphamide and the cyclophosphamide+buprenorphine groups (Pb0.05). (B) When the scores were tabulated togetherinto the combined distress scores, the cyclophosphamide group was signifi-cantly lower than the other three treatment groups (Pb0.05). In (A) and (B),each group represents the average of at least 8 animals.

Fig. 4. (A) Measurements of proteinuria inMRL/lprmice. Femalemicewere treatedwith buprenorphine, cyclophosphamide, cyclophosphamide+buprenorphine oruntreated controls for 10 weeks beginning at 11 weeks of age. Mice were analyzedfor proteinuria production by dipstick measurements weekly. (B) Measurement ofanti-dsDNA production at 11, 15 and 19 weeks of age. Sera were serially diluted 1:2beginning at 1:000 dilutions. Optical density (OD) readings are the average fromsera fromeachmouse performed in triplicate. Each graph represents the average of atleast 8 animals (⁎Pb0.05).


2.5.5. Combined distress scoresThe scores on each animal from the above categories (body

condition scoring, mentation, characterization of skin lesions,and other common indicators of stress) were added together togive a combined distress score.

2.5.6. LymphadenopathyDuring the physical exam, each mouse was palpated to

determine the extent of lymph node enlargement that waspresent. Lymph node enlargement was scored as follows: 0 =none, 1 = mild enlargement (palpable, but not easily visible), 2 =moderate enlargement (easily visible, but not interfering withmobility), and 3 = severe enlargement (easily visible andinterfering with mobility regardless of extent of interference).

2.6. Urine protein measurements

Urine was collected weekly and protein levels weremeasured using reagent strips. Urine protein levels werereported as negative, trace, 30 mg/dl (1+), 100 mg/dl (2+), or300 mg/dl (3+).

2.7. Sera collection and anti-dsDNA antibody levels

At 11, 15, and 18 weeks of age, the mice were anesthetizedwith isoflurane and bled from the retro-orbital sinus. At eachtime point, anti-dsDNA antibody levels were measured using anELISA as previously described (Reilly et al., 2006).

2.8. Flow cytometric analysis

At the time of sacrifice, themicewereweighed and necropsied.The spleens were removed, weighed, aseptically dissociated, andtreated with Tris–ammonium chloride–lysis buffer (pH 7.2) toremove erythrocytes. Flow cytometric analysis was performedusing fluorescein-isothiocyanate (FITC)-conjugated CD11c,CD21, CD62L (L-selectin), CD79b, and GL-7; R-phycoerythrin(R-PE)-conjugated CD3, CD4, CD23, CD138; PerCP-CY5.5-conjugated CD25, and Texas-Red-conjugated CD45R/B220 ratanti-mouse monoclonal antibodies (BD Pharmingen, San Diego,CA). Cells were resuspended in 0.1% BSA in PBS and aliquoted(0.25×106 cells/100 μl/well) into 96-well plates. One hundred μlof PBS including relevant monoclonal antibody equivalent to1 μg/1×106 cells added onto cells. The cells were incubated for30 min, at 4 °C. After 30 min, cells were washed with 0.1% BSAin PBS, and analyzed by flow cytometry (Coulter Epics XL/MXL, Hialeah, FL). For analysis, the cells were gated in forwardscatter versus side scatter. The CD4CD25CD62L cells werefurther gated including the CD25+ versus CD62L+ T cell subsetsover CD4+ T cells.

2.9. Kidney pathology

At the time of necropsy, the kidneys were removed anddivided into sections. One portion was placed in buffered


formalin for subsequent embedding in paraffin, followed bysectioning, H&E and PAS staining. Sections were assessed vialight microscopy for glomerular proliferation, glomerularinflammation, glomerular size, number of nuclei per glomeru-lus, crescents, necrosis and fibrosis. Each of these parameterswas graded from 0 to 3+ and an overall glomerular scoredderived. The pathology and morphometeric analysis wereperformed by a pathologist blinded to the groups (Dr. ArbenSanto).

2.10. Statistical analysis

Analysis of variance (ANOVA) was used to evaluatestatistical significance. A two-tailed P value of b0.05 wasconsidered significant.

Fig. 5. Renal pathology of MRL/lpr mice at 21 weeks of age. Representativecyclophosphamide+buprenorphine, (C) buprenorphine or (D) untreated controls. (cyclophosphamide+buprenorphine mice had significantly less renal pathology than

3. Results

3.1. Observation of animals

AsMRL/lpr mice age, they develop typical manifestations ofautoimmune disease similar to that seen in lupus patients,including alopecia, pruritus, and lymphadenopathy (Fig. 1A).When the scores for individual mice were tabulated, we foundno significant differences between the cyclophosphamide,cyclophosphamide+buprenorphine, buprenorphine, or controlgroups in the following parameters: characterization of skinlesions (Fig. 1B), other indicators of distress (Fig. 2A), bodycondition scoring (data not shown), or lymphadenopathy(Fig. 2B). Although there was no statistical difference betweengroups in these four parameters, there was a steady trend seen in

picture of a kidney from mouse treated with (A) cyclophosphamide, (B)E) Graphical tabulation of renal score pathology; the cyclophosphamide andthe buprenorphine or control animals (Pb0.05).

Fig. 6. Splenic weight measurements in MRL/lpr mice at 21 weeks of age.Female mice were treated with buprenorphine, cyclophosphamide, cyclophos-phamide+buprenorphine for 10 weeks beginning at 11 weeks of age. Controlsreceived no treatment. (A) Graphical representation of spleen weights to bodyweight ratio. (B) Spleen weight in grams and (C) body weight measurements ingrams. The cyclophosphamide and cyclophosphamide+buprenorphine micehad significantly smaller spleens and spleen to body weight ratios than thebuprenorphine or control animals (Pb0.05).


the data towards higher pain and distress scores regardingcharacterization of skin lesions, other indicators of distress, andbody condition scoring in the buprenorphine group and controlgroup when comparing to the cyclophosphamide or thecyclophosphamide+buprenorphine group.

For mentation, there was no significant difference between thecyclophosphamide and the cyclophosphamide+buprenorphinegroups. Likewise, there was no significant difference between thebuprenorphine and the control groups. Pain and distress, asindicated by mentation status, were significantly higher in thebuprenorphine and control groups when compared with either thecyclophosphamide or cyclophosphamide+buprenorphine groups(Fig. 3A).

When the scoring systems were tabulated together to form thecombined distress score, there was no significant differencebetween the buprenorphine group and the control group. Thecombined distress scores for the cyclophosphamide group weresignificantly lower than the remaining three study groups,including the cyclophosphamide+buprenorphine group. How-ever, the combined distress scores for the cyclophosphamide+buprenorphine group were also significantly lower than either thebuprenorphine or control groups, which had the highest scores forcombined distress out of the four study groups (Fig. 3B).

3.2. Sera and urine disease indices

As the mice aged, we measured common markers indicatingthe progression of lupus nephritis including proteinuria and thepresence of antibodies to double stranded DNA (Fig. 4). Theamount of protein in the urine was low in all groups butincreased as the mice aged (Fig. 4A). By 17 weeks of age, thecyclophosphamide and cyclophosphamide+buprenorphinegroups showed lower amounts of protein in the urine thaneither the buprenorphine or the control group (Pb0.05).

As MRL/lpr mice age, there is an increase in antibody levelsto double stranded DNA. The cyclophosphamide and cyclo-phosphamide+buprenorphine groups had significantly lowerantibodies to double stranded DNA in the sera than thebuprenorphine group or the control group (Fig. 4B). Theseresults showed that buprenorphine did not effect antibodyproduction.

3.3. Renal pathology

To determine the effects of buprenorphine on kidneyfunction, renal tissue was assessed from each mouse followingnecropsy (Fig. 5). Buprenorphine and control mice showedtypical glomerulonephritis, characterized by enlarged glomer-uli, proliferation of glomerular cells, infiltration inflammatorycells, increased mesangial matrix and cresent formation(Fig. 5A, B). In contrast, mice treated with cyclophosphamideor cyclophosphamide+buprenorphine showed a lesser degreeof glomerulonephritis, varying degrees of focal glomerular cellproliferation, occasional inflammatory cells, and a only a slightincrease in the mesangial matrix (Fig. 5C, D). There was also asubstantial reduction in the number of inflammatory cells in thecyclophosphamide and the cyclophosphamide+buprenorphine

groups when compared with either the buprenorphine or thecontrol groups, indicating that buprenorphine treatment did notalter renal pathology. When the scores were tabulated, thecyclophosphamide and the cyclophosphamide+buprenorphinegroups had significantly less renal disease than the buprenor-phine group or control group (Fig. 5E).


3.4. Spleen pathology

To determine the effect of buprenorphine treatment onsplenomegaly in MRL/lpr mice, the total body weight andspleen weight were measured in the mice at 21 weeks of age(Fig. 6). Spleen weights and spleen to body weight ratios weresignificantly lower in the cyclophosphamide and cyclophos-phamide+buprenorphine groups compared to the buprenor-phine group or the control group (Fig. 6A, B). There was nosignificant difference in body weights in any of the treatmentgroups (Fig. 6C). Splenomegaly was significantly reduced inthe cyclophosphamide and the cyclophosphamide+buprenor-phine group compared to the buprenorphine and the controlmice.

To further analyze splenic profiles, flow cytometricanalysis was performed (Fig. 7). We sought to characterizethe various T cell and B cell populations in the four studygroups. CD4+ CD25+ regulatory T cells (Tregs), constitute 5–10% of peripheral CD4+ T cells in normal naive mice andhumans. The subpopulation of CD4+CD25+CD62L+ cellspossesses a significantly higher suppressive capacity than theCD4+CD25+CD62L− T cells (Szanya et al., 2002). Percen-tages of CD4+CD25+ T cells and CD4+CD25+CD62L+ T cellswere slightly (but statistically significant) higher in the

Fig. 7. Flow cytometric analysis of splenic T and B cell activation markers incyclophosphamide, cyclophosphamide+buprenorphine, buprenorphine onlytreated, or untreated control MRL/lpr mice. (A) Splenic CD4+ T cells weregated for CD25 and CD62L analysis; (B) Splenic cells were analyzed fordifferent B cell surface markers. Data represents the mean±standard deviationof at least five mice at 21 week of age.

cyclophosphamide group compared to the control group(Fig. 7A). The percentages of these two groups were alsohigher in the cyclophosphamide+buprenorphine group andthe buprenorphine group when compared to controls, but thedifference was not statistically significant.

We also examined the different B cell populations in the fourstudy groups. The percentages of germinal center B cells(CD45R/B220/CD3/GL-7), marginal zone cells (CD45R/B220/CD23/CD21), and dendritic cells (CD45R/B220/CD11c/Ly6c)were not different between cells from the four study groups. Theplasma cell (CD45R/B220/CD138/CD79b) population wasslightly higher in the control group (Fig. 7B).

4. Discussion

The use of MRL/lpr mice has been a widely accepted animalmodel to study SLE for over 30 years. Although the micedevelop disease manifestations similar to humans, concernsexist as to the humane use of older mice since as they age themice develop severe skin and ear pathologies. Therefore, thesestudies were conducted to examine if an analgesic could beadministered to the mice to alleviate the skin manifestationswithout interfering with autoimmune disease. Buprenorphinewas chosen due to the low incidence of tolerance development,its potency as an analgesic, the long duration of action, the watersoluble nature of the compound, and its reported activity inrodent models, including its effects on the immune system.

An important consideration when using opioid analgesics forchronic administration is the development of tolerance.Tolerance refers to the diminution in the response of a patientto a drug after prolonged use. Regarding tolerance developmentto opioids, as opioid use becomes prolonged a larger amount ofdrug is required to reach the same plane of analgesia that wasreached with the initial dose. Although buprenorphine can causethe development of tolerance, it is thought to be associated withless severe tolerance when compared with other opioids.Tolerance was not one of the parameters evaluated in thisstudy; however, the decreased incidence of tolerance develop-ment with buprenorphine compared to other opioids was aconsideration for choosing buprenorphine as the most appro-priate analgesic for chronic administration. A study by Gringuazet al. showed tolerance to buprenorphine developed in micefollowing 14 days of treatment at 1.2 mg/kg given s.c. b.i.d.,however the tolerance that developed was the least noticeablewith buprenorphine when compared to the other drugs tested,including butorphanol, nalbuphine, and cyclorphan (Gringauzet al., 2001).

Concerning the effects of buprenorphine on the immunesystem, Banerjee and Sarkar reported that buprenorphineadministration altered immune function in mice by decreasinglymphocyte and monocyte counts and increasing neutrophilcounts. However, these effects were transient and resolvedwithin 45 days of drug withdrawal (Banerjee and Sarkar, 1997).In another study, buprenorphine was shown to have nosignificant effect on immunity based measurements of lympho-proliferation, natural killer cell activity, interferon-gammaproduction, and interleukin-2 production (Martucci et al.,


2004). Other studies have also indicated that buprenorphine hasminimal long term effects on the immune system (D'Elia et al.,2003; Gomez-Flores and Weber, 2000; Lindsay et al., 2005) anddoes not alter renal perfusion in a rodent model of renalischemia (Deng et al., 2000).

Evaluating pain and distress in laboratory animals can bechallenging. Based on guidelines from the Institute ofLaboratory Animal Research, distress can be defined as thestate in which an individual is unable to adapt to increasing orpersistent stress which results in maladaptive behaviors.Examples of maladaptive behaviors include inappropriate socialinteractions, self-mutilation, and repetitive stereotypic move-ments. When initially presented with a stressor, an animal willeither adapt to the stress or will develop maladaptive behaviorsthat will lead to distress. Pain, by comparison, is considered tobe a stressor and therefore one of the causes of distress. Some ofthe common indicators of pain and distress in rodent specieshave been identified as the following: abnormal vocalizations,self-mutilation, changes in normal group behavior or grooming,depression as indicated by a reluctance to move and decreasedactivity, abnormal appearance including hunched positioning orruffled hair coats, or increased respiratory rate and associatedgrunting or chattering on expiration (Institute of LaboratoryAnimal Resources (U.S.). Committee on Pain and Distress inLaboratory Animals, 1992).

This study was designed to determine if the use of ananalgesic would aid in the relief of distress without having aneffect on autoimmune disease progression in the MRL/lprmodel. Concerning the parameters measured, the distinguishingfactor was whether or not the parameter could be altered bytreatment with analgesics. For example, any parameter that wassuspected to be unaffected by administration of analgesics, butwould be changed by progression of disease was used toindicate autoimmune disease progression. These objectivemeasures were presence or absence of proteinuria, auto-antibody production, renal involvement, splenomegaly, spleno-cyte profiles, and the development of lymphadenopathy. Ourhypothesis was that these parameters would not be affectedbetween the buprenorphine-treated groups and the non-buprenorphine-treated groups. If this hypothesis was correct,then buprenorphine could be used as an analgesic withoutinterfering with common parameters used to indicate autoim-mune disease progression.

In this study, there was no evidence to suggest thatbuprenorphine negatively impacted the progression of autoim-mune disease in MRL/lpr mice. In all of the parameters used toindicate disease progression, there was no significant differencebetween the buprenorphine and control groups, or between thecyclophosphamide and cyclophosphamide+buprenorphinegroups. This indicates that the administration of buprenorphinedoes not interfere with the progression of autoimmune diseasein MRL/lpr mice.

Parameters used as indicators of distress in this studyincluded body condition scores, changes in mentation, severityof skin lesions, abnormal respiratory rate, presence of ruffledhair coats indicating a lack of grooming, huddling behaviors,and pruritus. Two of these parameters, skin lesions and pruritus,

have been used as measures of autoimmune disease progressionin other studies. However, analgesic administration couldinterfere with the measurement of these two parameters. Forexample, depending on the underlying cause of pruritus,scratching behavior can be either a cause of pain or a responseto pain. If the pruritus seen in MRL/lpr mice is associated withpain that occurs secondary to self-mutilation, then theadministration of analgesics could decrease the incidence ofpruritus. However, if the pruritus is associated with diseaseprogression and acts only as a cause of pain, analgesicadministration would be expected to have no effect on theincidence of pruritus. The severity of the skin lesions seen canbe considered in a similar manner. If the lesions develop as aresponse to pain, analgesic administration would be expected todecrease the incidence of skin lesions whereas if the lesionsdevelop solely due to disease progression, then analgesicswould be expected to have no effect. Since the measurement ofthese two parameters could be influenced by analgesicadministration, they were not appropriate indicators of diseaseprogression in this particular study. Therefore, these twoparameters were used as indicators of pain and distress ratherthan as indicators of disease progression.

Based on measurements of the parameters used to indicatedistress, this study demonstrated that buprenorphine, whendosed at the previously published dose of 0.09 mg/kg per mouseper day in the drinking water (Lindsay et al., 2005), was notadequate as the sole method of alleviating pain and distress inMRL/lpr mice that have concurrent evidence of autoimmunedisease. This was indicated by the lack of decrease in distressscores between the groups treated with buprenorphine and thegroups not treated with analgesics.

There are several likely explanations for this finding. Onepotential consideration is the dose of buprenorphine and theroute of administration. Although the dose of buprenorphineused in this study has been cited as being effective (Lindsayet al., 2005), there has been very few pharmacologic trials todetermine the most appropriate dose of buprenorphine foranalgesia in mice. Buprenorphine is typically used to treat mild,moderate, and severe pain by varying the dose given (Christophet al., 2005). Dose ranges for buprenorphine vary from0.002 mg/kg to 0.5 mg/kg (Christoph et al., 2005; Gadeset al., 2000; Lizasoain et al., 1991; Mizoguchi et al., 2003; vanLoo et al., 1997). The wide range of doses indicates the lack ofcontrolled studies involving buprenorphine in mice. However,the dose used in this study (0.09 mg/kg/day) falls within theexpected therapeutic dose range (Banerjee and Sarkar, 1997;Christoph et al., 2005; Gades et al., 2000; Gross et al., 2003;Lindsay et al., 2005; van Loo et al., 1997).

It is also possible that the administration of buprenorphine inthe drinking water may affect the absorption or efficacy of thedrug. However, buprenorphine has been demonstrated to beeffectively absorbed via administration in the drinking water andprovided appropriate analgesic efficacy in rats (Cooper et al.,1997). Due to the close phylogenetic relationship between miceand rats, it is likely that absorption and efficacy of buprenorphineadministration in the drinking water of mice would be similar tothat seen in rats. Furthermore, Lindsay and colleagues have


shown that buprenorphine administered via the drinking water,at the same dose as was used in this study, decreased pain anddistress in an experimental murine model of T. gondii (Lindsayet al., 2005). Because of the lack of pharmacological studies todetermine the most appropriate therapeutic range of buprenor-phine in mice when administered via drinking water, it isdifficult to say whether buprenorphine at a higher dose wouldprovide adequate analgesia to decrease pain and distress inMLR/lpr mice.

A second potential explanation for our findings involves theunderlying cause of distress in autoimmune skin diseases.Recently published findings have indicated that spontaneousscratching behavior in MLR/lpr mice may be a response toautoimmune pruritus rather than as a response to pain or distress(Umeuchi et al., 2005). This would suggest that controlling thepain in these animals would not decrease the underlying cause ofthe scratching, the autoimmune pruritus. In a normal animal, thenatural negative feedback mechanism for excessive scratchingbehavior includes initiating a painful response associated withthe self-trauma. This painful response inhibits further scratching.Therefore, it could be hypothesized that analgesic administrationwould decrease the pain sensation caused by self-trauma andpotentially disrupt the normal negative feedback loop. As aresult, interruption of this feedback loop could cause worseningself-trauma in animals treated with analgesics if the underlyingcause of the pruritus was not controlled.

Another explanation for the lack of desired effect ofbuprenorphine could be attributed to its pharmacologicalproperties. Currently, there are four opioid receptors that arethought to be clinically important. Exogenous opioids havevariable affinities for these four receptors, resulting in thediffering effects of opioid drugs. These four receptors includethe three classical opioid receptors (μ, κ, and δ) which areprimarily involved in the analgesic response to opioids, and theopioid receptor-like receptor ORL-1 which is thought to opposethe analgesic effect seen with stimulation of the classical opioidreceptors. Exogenous opioids are either agonistic, partialagonistic, or antagonistic at these receptors. Buprenorphine isconsidered to be an agonist–antagonist. It is a partial agonist atthe μ and ORL-1 receptors and an antagonist at the κ and δreceptors. The desired analgesic effects of buprenorphine areassociated with its actions as a μ agonist, while its relative lackof negative side effects when compared to other opioids isthought to be due to its antagonistic properties at the κ and δreceptors as well as its agonistic properties at the ORL-1receptors which moderate the analgesic effect (Christoph et al.,2005). In addition to their role in analgesia, central μ receptorshave been found to play a role in the processing of the itchsensation when using a pruritogen-induced scratching model inmice, while κ receptors are thought to suppress this itchsensation (Umeuchi et al., 2003). Therefore, while the actions atthe μ receptors may be causing sufficient analgesia, stimulationof these receptors may also be assisting in the processing of theitch sensation. As a κ antagonist, buprenorphine would beunlikely to contribute to suppression of this itch sensation asseen with κ agonists. An example of a κ agonist that hasrecently been shown to suppress the itch sensation is nalfurafine

(Umeuchi et al., 2005). Taken together, the contribution ofbuprenorphine to the itch cycle could overwhelm the benefits ofits analgesic properties.

In conclusion, buprenorphine should still be considered as apotential analgesic of choice for reducing pain and distress inmice. It can also be considered as a compound that does notimpact the progression of autoimmune disease in MRL/lprstrains when given in the drinking water at 0.09 mg/kg/mouse/day, based on the criteria measured in this study. However, itspotential use to moderate scratching behavior and/or serve as ananalgesic for MLR/lpr mice has yet to be substantiated. Furtherstudies are needed to determine if buprenorphine, and opioids ingeneral, can be used effectively to prevent pain and distressassociated with the progression of disease in mice sufferinglupus-like syndromes.

Acknowledgements

The authors would like to thank the staff of the Teaching andResearch Care and Support Service at Virginia Tech for theirassistance with this project as well as The Arthritis Foundationfor their gracious support of Dr. Christopher Reilly's research.

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clinical efficacy of buprenorphine to minimize distress in mrl/lpr mice

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