The uroprotection of mesna on cyclophosphamide cystitis in rats. Its consequences on behaviour and brain activities

0 Academic

des sciences

Pharmacology

/ Pharmacologic

/ Elsevier,

Paris

The uroprotection of mesna on cyclophosphamide cystitis in rats. Its consequences on behaviour and brain activities L ‘action woprotecttice du mesna SW la cystite au cyclopbospbamide cbez le rat. Sesconst?quencessuf- le compotiement et l’activitk nerveuse Daniel

Men6treya*,

Karine

Bona, Jean-FranGois

Michielsb,

Wnite 161 de I’lnserm, 2, rue d’Al&ia, 75014 Paris, France bLaboratoire d’anatomo-pathologie, centre hospitalo-universitaire =Departement Douleur, centre hospitalo-universitaire de Nice, (Received Note

28 September

communicated

1998;

by Pierre

accepted

25 February

Michel

Lantkri-Minet”!

de Nice, 06002 Nice, France hopital Pasteur, 06002 Nice, France

1999)

Buser

Abstract - We studied the uroprotective effect of mesna, at doses of 40-300 mg/kg/i.p., in single or fractioned injections, on the development of cyclophosphamide (CP, 100 mg/kg/i.p.) cystitis in rats. The study concerns the histological, behavioural and nervous aspects of the disease. The specific effects of mesna, when injected alone, have also been considered. The mesna itself does not have specific deleterious effects, except at a dose of 300 mg/kg which provokes a moderate vesical inflammation although without consequence on the animal’s behaviour. Mesna offers good protection against CP cystitis for only certain posologies. The uroprotective effects of mesna reach maxima at doses of 40-l 00 mg/kg and for fractioned injections given over the entire time frame of the urinary toxic release. The uroprotective effects of other posologies are only partial. The nervous activities were studied through the expression of Fos protein. The repetitive intraperitoneal injection of mesna induced a spinal activity and a preferential contralateral activity of the trigemino/reticular areas of the brainstem spinal cord junction - an effect which was reduced in the presence of CP. The prevention of cystitis by mesna was accompanied only by a reduction in spinal Fos activity, the supraspinal activities remaining high and in strict relationship with the vagal afferent activity. In conclusion, the uroprotective effect of mesna, which requires appropriate posologies, has led to the confirmation of the spinal actions of the CP cystitis, probably via the pelvic nerve, but did not allow a clear distinction between the consequences of the systemic (vagal) and local (spinal, pelvic) actions of CP at supraspinal level. 0 Academic des Sciences / Elsevier, Paris visceronociception

I spinal cord / vagal nerve / bladder

I nervous system

R&ume - Nous avons 6tudii: l’effet uroprotecteur du mesna, aux doses de 40 2 300 mg/kg/i.p., en injections unique ou fractionnkes, sur le developpement d’une cystite, induite par le cyclophosphamide (CP, 100 mg/kg/i.p.) chez le rat. L’6tude concerne les aspects histologiques, comportementaux et nerveux de la maladie.

* Correspondence

and reprints:

C. R, Acad. Sci. Paris, Sciences 1999,322,505-515

[email protected] de la vie / Life Sciences

c

D. Menetrey

et al.

Les effets propres du mesna, lorsqu’il est inject6 seul, ont aussi 6t6 consid&&. Le mesna seul n’a pas d’effets dUt&es propres, sauf 2 la dose de 300 mg/kg, pour laquelle il provoque une 16gGre inflammation v&iicale mais saris repercussions comportementales. Le mesna ne protgge efficacement de la cystite au CP que pour certaines posologies. Ses effets uroprotecteurs sont maximaux aux doses de 40 2 100 mg/kg et pour des injections fractionnkes, 6chelonn6es sur toute la p6riode d’excrktion des toxines urinaires. Les effets uroprotecteurs des autres posologies ne sont que partiels. Les activites nerveuses ont et6 etudikes par l’expression de la protkine Fos. L’injection intrapkritonkale repWe de mesna induit une activite spinale et preferentiellement contralatkrale des regions trig6mino/r&iculaires de la jonction bulbospinale, effet qui se reduit en pr&sence de CP. La prkvention de la cystite par le mesna ne s’accompagne que d’une reduction du marquage Fos spinal, le marquage supraspinal restant 6lev6 et en relation 6troite avec une forte activitC vagale affkrente. En conclusion il a &Z demontre que l’effet uroprotecteur du mesna, qui requiert des posologies bien adaptkes, permet de confirmer les actions spinales, d’origine pelviennes, de la cystite au CP, saris cependant permettre de clairement distinguer, au niveau supraspinal, entre les cons& quences nerveuses des effets systCmiques (vagaux) et locaux (vessie, pelviens) que le CP engendre. 0 Acadkmie des Sciences / Elsevier, Paris visc&onociception

Version

/ moelle

CpinPre

/ nerf

vague

/ vessie

/ syst*me

abri?g&

Les cystites constituent un groupe de maladies inflammatoires souvent t&s douloureuses et invalidantes. Pour etudier leur rt5percussion sur l’activitk du systGme nerveux central, nous avons utilis@ le modgle de cystite au cyclophosphamide (CP) qui reproduit chez l’animal une situation pathologique rencontree en clinique humaine. Le CP est un agent antitumoral dont les m&abolites urinaires, essentiellement l’acrolCine, engendrent une cystite 5 Cvolution progressive par contact direct avec les parois vksicales dont elle fragilise 1’6pith6lium. En dose unique de 100 mg/kg, inject6 par voie intrap&itoneale, le CP dkclenche chez le rat une cystite devenant hkmorragique en 4 h. Cette cystite, identifiable au niveau histologique, s’accompagne de variations comportementales et engendre des activites nerveuses, de type Fos et Krox-24 positives. La cystite au CP est un modele particulierement approprie 2 l’etude des mecanismes g&&ateurs de douleurs vis&rales. L’ktude presentee ici concerne l’effet uroprotecteur du mesna au regard de ce type de cystite. Le mesna constitue un antidote specifique de l’acro&ne, le metabolite urinaire le plus toxique du CP. Le mesna est instable en milieu sanguin. 11y est spontanCment oxid en un dim&e inactif, le dimesna, 5 partir duquel il sera r6gCn&Z au niveau renal. Le dimesna est une substance totalement inerte. I1 n’a aucune action syst@mique et n’interfire ni avec le CP ni avec ses mktabolites, hepatiques ou renaux. Le mesna, une fois r&g&&6 P partir du dimesna et lib&6 localement par filtration glomerulaire, va detoxifier l’acrol6ine en s’y complexant pour former une substance qui, physiologiquement inerte, ne presentera plus d’agressivitg pour la muqueuse v6sicale. Le mesna a &t6 inject6 aux doses totales de 40 5 300 mg/kg/i.p. Les injections ont et6 faites en doses unique ou bien fraction&es par quarts et espackes de 1 h. Cette seconde procedure permet de compenser la clairance rapide du produit et done de mieux adapter sa cinetique d’excretion 5 celle des toxines urinaires. L’injection unique de mesna et la

506

nerveux

premi&e injection des doses fractionn6es suivent immediatement une injection initiale de soluti‘ salin, quand il s’agit d’etudier les effets propres du mesna, ou de CP, quand il s’agit d’Ctudier ses effets uroprotecteurs; les trois injections fraction&es suivantes sont effect&es au rythme d’une par heure. Au total, 73 animaux ont et6 consid&& et observes sur une periode de 4 h suivant l’injection initiale, ce laps de temps ttant la du&e minimale pour qu’une cystite se developpe completement apr&s une injection unique de CP. Les observations ont tt6 confrontees 2 celles provenant de douze animaux n’ayant regu que du solutC salin dans des conditions d’injection identiques. L’ktude concerne les manifestations histologiques, comportementales et nerveuses de la maladie, les activites nerveuses &ant 6tudi6es au travers de l’expression de la proteine Fos. Les r&ultats histologiques et comportementaux demontrent que le mesna, d’une part, n’a aucun effet del&re propre, sauf 5 la dose de 300 mg/kg pour laquelle il provoque une leg&e inflammation v$sicale mais sans r@percussions comportementales, et, d’autre part, ne prot?ge eKcacement de la cystite au CP que pour certaines posologies. Ses effets uroprotecteurs sont maximaux aux doses de 40 5 100 mg/kg et pour des injections fraction&es. Les effets uroprotecteurs des autres posologies ne sont que partiels. L’efflcacite uroprotectrice du mesna en injection unique reste done partielle comparee 5 celle d’injections fraction&es r6pMes. L’association CP et mesna, quand il est administre 2 la dose totale de 300 mg/kg et en doses fractionnees, gCn6re une cystite modWe s’accompagnant de modifications comportementales. L’expression de la protkine Fos demontre que I’injection intrap&itonCale rCp@e de mesna induit par ellememe des activites spinale et trig6minor6ticuiaire 5 la jonction tronc c&&bral-moelle CpiniPre. L’activitk spinale concerne essentiellement les couches superficielles de la come dorsale et la couche X ainsi que son extension caudale, la commissure grise dorsale sacree. L’activite supraspinale est 5 p&dominance contralaterale aux sites d’injection. Les strucC. R. Acad.

Sci. Paris, Sciences

de la vie / Life Sciences 1999,322,505-515

Mesno,

tures concernkes sont les noyaux rkticulaire intermediaire caudal et dorsal, les groupes catkholaminergiques Al/Cl et la partie adjacente du noyau spinal du complexe trigkminal. Cette derniere rkgion, la plus ventrale du noyau, correspond 2 la zone de transition entre les subdivisions trigeminales caudale et interpolaire et inclut le noyau paratrigeminal ventral. Cet effet qui est topographiquement organis& et engendre par l’injection intrapkitonkale est done t&s probablement d’origine locale. Une telle activiti: ne peut @tre dkcelke en prkence de CP, ce qui laisse supposer qu’elle reste sous le contrble des influx vagaux que l’injection de cette substance engendre (inhibition d’origine vagale). Cette rCgion trigkmino-rkticulaire est 5 tri’s fort caractke convergent (somatique, visckal, trigeminal, autonomique) et pourrait faire partie du substrat anatomique oti les mimiques faciales, les reflexes lacrymaux et les ajustements autonomiques (hkmodynamiques, respiratoires) sont klaborits en r& ponse 5 des stimulations nociceptives. L’inhibition d’origine vagale qui s’y exerce pourrait s’apparenter aux effets inhibiteurs que les affirences vagales exercent sur la transmission nociceptive et dont la demonstration 5 dkjk et6 faite par le passe. L’injection de CP engendre une activitk Fos renforcCe dans la colonne parasympathique lombosacrke. Des activitk importantes peuvent aussi @tre observkes dans le noyau .

1. Introduction

tumour agent currently toxicity of which comes byproducts, especially

used in clinics [31, the bladder from the release of toxic renal acrolein [4, 51. The prolonged

contact of these final byproducts with the bladder wall during urine accumulation and retention generates cystitis. Using this model it was demonstrated that CP cystitis genesis results in behavioural modifications along with differential activation of certain areas in spinal [l], brainstern [6, 71 and telencephalic structures [81 but with minor effects at mesodiencephalic levels 191. Spinal activities concern lamina 1 of the dorsal horn, lamina X including its sacral extention the dorsal gray commissure and the sacral parasympathetic column. Brainstem activities concern the dorsal vagal complex, the ventrocaudal bulbar reticular formation and the dorsal paratrigeminal nucleus. Telencephalic activities concern both the bed nucleus of the stria terminalis in the dorsal part of its lateral division and, to a lesser degree, the nucleus centralis of the amygdala mostly in its caudal portion. Because of the common use of CP in the curative treatment of human tumours, ample efforts have been made to develop agents having a uroprotective action without interfering with the beneficial chemotherapeutic effect. To do so, chemicals must remain in the intravasal space, without

i.e. without having

penetrating into the tissues; intrinsic toxicity or systemic

CR. Acad. Sci. Paris, Sciences 1999.322.505-515

de la vie / Life Sciences

be inert, i.e. interaction

and

cystitis

paratrigkminal dorsal, une zone de projection largement spkifique des affirences primaires du nerf vague, le noyau sensitif du nerf vague et la plupart des regions 1 composante autonomique qui lui sont associkes (formation kticulke bulbaire ventrolatkrale, locus coeruleus et regions avoisinantes, noyau 0 et aire pkiaqueducale pars alpha, aire parabrachiale, noyau central de l’amygdale, noyau du lit de la strie terminale). La prkvention de la cystite au CP par le mesna ne s’accompagne que de la reduction du marquage Fos spinal de la colonne parasympathique lombosacrke. Cette activitk est done bien li6e & la ge&se de la cystite elle-m&me. Le marquage supraspinal reste en revanche 6lev6 et en liaison Ctroite avec l’activation du systeme affirent vagal comme le dkmontre I’activitk du noyau paratrigkminal dorsal. Ces activitCs doivent done &tre considMes, pour une part, comme provenant des effets systemiques que le CP induit plutBt que par la cystite elle-m@me. En conclusion il a 6% dCmontr6 que l’effet uroprotecteur du mesna, qui requiert des posologies bien adaptees, permet de confirmer les actions spinales, d’origine pelvienne, de la cystite sans cependant permettre de clairement distinguer, au niveau supraspinal, entre les conskquences nerveuses des effets systkmiques (vagaux) et locaux (vessie, pelviens) que le CP engendre

with

Cystitis induced by cyclophosphamide (CP) has recently been proposed and used as a model for studying visceral pain in freely behaving rats [I, 21. CP is an anti-

cyclophosphamide

blood

circulating

substances;

act

locally

and

specifi-

cally at the renal level, i.e. preventing the release of toxic byproducts or detoxifying them. Both clinical and experimental observations [I O-l 31 have shown that one of these agents is mesna (sodium-2-mercapto-ethane sulphonate). The mechanism of the uroprotective efficacy of mesna is based on the formation of non-toxic complexes with the renal CP byproducts. Following administration, mesna is rapidly and spontaneously oxidized in blood plasma to a physiologically inert disulphide form, dimesna, the reduction of which during glomerular filtration will partly regenerate mesna, the reactive form. If toxic CP byproducts are present, mesna will complex them specifically at the renal level, thus preventing both their release and the development of the associated cystitis. In the present paper we analysed the uroprotective action of mesna on CP-cystitis and its repercussion on the behavioural and brain activities which are associated with cystitis genesis. The specific effects red.

of mesna,

2. Materials

when

injected

alone,

were

also

conside-

and methods

Experiments were performed on male Sprague-Dawley rats (Centre d’elevage Charles Rivers, France) housed in cages with sawdust bedding, given food and water ad libitum and kept in an animal house at a constant temperature of 22 “C with a 12-h alternating light-dark cycle. The experiments were performed on naturally behaving, awake animals and conformed to Principles of laboratory animal

care (NIH

publication

86-23,

revised

1985)

and

507

D. Menetrey

et al.

ethical principles ted, animals were lowed to behave med during the these animals. 2.1.

[14]. One transfered naturally. light period,

Experimental

hour before experiments to individual cages All

experiments the normal

and

staral-

were perforsleep cycle for

shams) doses.

procedure

or saline + mesna (n = 28, mesna Data refering to the uroprotective

mesna against cystitis been initially injected instead of saline (12 ted with CP + mesna

of the five behaviours over these was expressed in seconds (up to 300 period) and multiplied by a factor behaviour’s apparent degree of di-

sease. The factor for normal behaviour was 1, 2 for ocular discharge, 3 for piloerection, 4 for rounded-back posture with alertness, 5 for rounded-back posture with immobility. Behavioural curves were obtained from these scores

The effects of mesna (Uromitexan) were tested in a range of 40-300 mg/kg total doses. Data refering to the proper effects of mesna were obtained from animals having been injected with either saline + saline (n = 12, saline various

The timing for each measurement periods per 5-min measurement associated with that

were obtained from with CP (Endoxan, received CP + saline, at various doses). All

shams) effects

at of

animals having 100 mg/kg/i.p.) 33 were injecinjections were

and expressed initial injection. 2.3.

Evaluation

The

bladder

as a function

of the

of bladder was

survival

time

after

the

inflammation

removed,

postfixed

(10%

formalin

for

3 days), and embedded in paraffin, then cut on a microtome into 5-pm-thick sections. Sections were mounted, cleared in toluene, hydrated in alcohol, stained with hae-

performed intraperitoneally on one side and in the inferior lateral quadrant of the abdomen, resulting in the administration of the same total volume of liquid (2 mL). The initial injections, either saline or CP, were of 1 mL volume. The subsequent injections, either saline or mesna (I mL total

matoxylin, eosin and safran. After final dehydration the sections were coverslipped with Eukit. Histological examination of the bladder was used to estimate the severity of lesions. The state of the bladder was assessed with a 4-grade scale in a blind setting, using chorionic oedema, fibrin deposit, epithelial thinning, desquamation, pe-

volume), injection

were was

techial haemorrhage first grade consisted

(saline or the initial injection clearance after the

CP); the series of four injections began just after injection and at 1 -h intervals each. The multiple procedure was chosen to compensate the rapid of mesna [lo-l 31. All animals survived for 4 h initial injection, i.e. the shortest postinjection

either single or in a series of four. performed immediately after the

The single initial one

and cell of animals

second grade, animals excessive submucosal corresponding to the response); third grade, with epithelial cleavage

infiltration with no

as criteria. The bladder lesion;

with simple oedema (presence of fluid due to exudation of plasma early features of the inflammatory animals with oedema associated and thinning resulting in mucosal

survival time for animals to develop complete cystitis a single CP injection [l 1. Behavioural reactions were died over the 4-h survival time period. Bladder state central nervous activities were studied at the end of the survival time period after the animals had been killed

after stuand 4-h by

abrasion, fibrin deposit and the beginning of a polymorphonuclear leucocyte infiltration as signs of mild cystitis; fourth grade, animals with complete cystitis corresponding to an increase in severity and spread of all the signs of cystitis described above, plus petechial haemorrhage. It

intracardiac der deep consisted

unand (PB)

was observed animals could

on rare display

and

as a result

perfusion. The perfusion was performed anaesthesia (40 mg/kg/i.p. of pentobarbital) of 200 mL of 0.1 M phosphate-buffered

saline (PBS, pH: 7.4) followed by 400 mL of 4% maldehyde in PBS. Behavioural, histological and studies were conducted in parallel. Uninjected served as controls. 2.2.

Evaluation

of behavioural

parafornervous animals

impairment

Behavioural reactions were quantified using the microcomputer program developed by Tarapacki and Kristal [I 51. The following five types of abnormal behaviours that cystitic animals can develop were considered: 1) ocular discharge (lacrimation) evidenced by chromodacryorrhoea, 2) piloerection, 3) assumption of a particular ‘rounded-back’ posture that was later accompanied by 4) head immobility and 5) various brief ‘crises’ (tail hyperextension, abdominal retractions, licking of the lower abdomen, backwards withdrawal movements). The analysis was performed on a series of 11 measurements (5 min each every 20 min for the total period of 4 h with the first measurement taken 30 min after the initial injection). Three rats, injected 45 min apart, were tested in sequence.

508

probably

2.4.

Evaluation

occasions that even control some signs of chorionic of the perfusion

of nervous

or sham oedema,

protocol.

activity

The neuraxis of animals was ted overnight in phosphate-buffered

removed 30%

and cryoprotecsucrose solution

at 4 “C before cutting. Frozen serial transverse sections (40 pm thick) were collected in PBS to be processed immunohistochemically as free-floating sections. Sections were incubated in 10% normal goat serum in PBST (PBS and 0.3% Triton X-100) for 30 min, followed by c-fos primary antiserum for 2 days at 4 “C. The antibody, a kind gift from Dr Hunt (MRC Centre, Cambridge, UK), was polyclonal. The properties of this antibody, which is directed against a synthetic peptide sequence (2-l 7) specific to the N-terminal portion of the Fos protein, have been previously reported [161. Highly diluted serum (l/l 5 000) was used to promote differential detection of stimulusevoked versus baseline expression. Although specificity controls (peptide absorption, omission of first antibody) were conclusive, staining must be considered as IEGencoded

protein-like

immunoreactivity.

C. R. Acad.

Sci. Paris, Sciences

After

incubation

de la vie / Life Sciences 1999,322,50.5-515

Mesna, in primary antiserum, ges of normal goat

sections serum (1%

tories). Following a final developed in a-naphthol tion [17]. The incubation

anti-rabbit serum, washed for 1 h in avidin-biotinElite from Vector Labora-

3.1.

wash in PB the sections were ammonium carbonate solumedium consisted of 89.5 mL

coverslipped with Eukit. Graded omitted before coverslipping.

2.5.

Treatment

dehydration

injection sites. Supraspinal labelling was seen at hindbrain (dorsal vagal complex [DVCI, ventrocaudal bulbar reticular formation [vcBRF], laterodorsal tegmental nucleus [LDTg], parabrachial area, locus coeruleus, nucleus 0 and central gray pars alpha), mesodiencephalic Westphal nucleus [EWI, intergeniculate leaf rior [PVP] and anterior [PVA] paraventricular

Table + mesna

are expressed on one side.

I. Mean values (k SE) of histopathological at a total dose of 300 mg/kg.

bladder

Groups Saline one four Saline one four All

Mean + saline injection injections + mesna injection injections

animals

similar to with 1 mL The series injection CR.

Acad.

were

was observed contralateral the caudal intermediate Al/Cl catecholaminergic

as the

diseases

bladder

in animals

having

been

injected

Fischer’s

state

(Edingerpostethalamic

to the injections and included reticular nucleus (clRN), the groups and the most ventral tip

intraperitoneally

PLSD

versus

with

corresponding

either

saline

saline

only

or saline

group

1.2 * 0.2 1 .l & 0.1 (300

mg/kg

total)

2.3 k 0.2 2.6 f 0.4 injected

with

the

same

amount

of liquid

(2 mL) but

P< 0.01 P < 0.004 following

different

protocols,

those used when studying the uroprotective effects of mesna on CP cystitis. All animals of either saline (saline shams) or mesna (300 mg/kg, mesna shams) solution, either once of injections started immediately after the initial injection of saline. The data are from of saline. Mesna at dose of 300 mg/kg had mild but definitive bladder toxicity. Sci. Paris,

1999,322,505515

[IGLI,

nuclei, hypothalamus, supraoptic nucleus) and telencephalic (nucleus centralis of amygdala, bed nucleus of stria terminalis, lateral septum) levels. Repetitive injections of mesna evoked labelling in the medulla oblongata at frontal levels passing through the midcaudal portion of the lateral reticular nucleus (LRN). Preponderant labelling

way ANOVA. The PLSD Fischer’s test was used to determine probability values between animal groups. All neuronal labelling was plotted with a camera lucida. Fospositive cells were counted on five spinal sections and for all sections passing through the supraspinal structures results section

and repetitive injections, behavioural consequendiseases remained quite

minor (figure 7). Saline-injected animals had consistent bilateral Fos staining at both spinal and supraspinal levels. Spinal staining concerned the superficial layers and neck of the dorsal horn as well as the dorsal gray commissure (DGC), this latter being predominantly contralateral to the

of data

study. Fos quantitative of positive cells per

in association with saline and at total 40 to 200 mg/kg, had no or very minor

0.01 and P < 0.004 for single respectively, (table 0. However, ces associated with these bladder

Data-collection phases were always performed by a single blind observer. Data are expressed as mean value + standard error (m f SE) and were analysed statistically (Statview 4.0, Abacus Concepts Inc., 1992) using a one-

under number

alone

fractioned (2.6 + 0.4) doses (table f). These values, which corresponded to pronounced chorionic oedema often associated with epithelial cleavage, significantly differed from those observed in corresponding saline shams (P <

slides, air dried and crystal violet [Aldrich,

alcohol

of mesna

respectively). No behavioural modifications were observed under these conditions. Mesna, injected at a total dose of 300 mg/kg, was slightly toxic. Similar effects were obtained when the drug was injected in single (2.3 f 0.2) or

no. 425551 solution in PB). Following two short rinses in distilled water to remove the excess of staining, sections were sequentially differentiated in 70 and 95% alcohol, the time of differentiation being evaluated under a microscope. At that time staining appears as an intense blue-violet colour. Sections were finally air dried, xylene treated and was

cystitis

effects on bladder aspect. Mean bladder states, which under these conditions were graded from 1.2 ? 0.2 to 1.6 + 0.2, did not significantly differ from those of saline shams (1.2 + 0.2 and 1 .l +O.l for single and repetitive injections,

dissolved. The incubation was performed at room temperature for 3 min with 0.03% hydrogen peroxide followed by two rinses of PB. The a-naphthol staining is a fine grey-violet precipitate which must be intensified and made alcohol resistant to be easily identified. Thus, secwere mounted on gelatin-coated enhanced (3 min in 0.025%

Effects

Mesna, injected doses ranging from

PB, 10 mL ammonium carbonate solution (1% in distilled water), 0.5 mL a-naphthol solution (N-199-2 Aldrich, 10% in absolute alcohol), prepared with vigourous stirring as it takes several minutes for a-naphthol to be completely

tions dye

and

3. Results

were washed in three chanin PEST), incubated at 4 “C

overnight in biotinylated goat twice in PB and then incubated peroxidase complex (Vectastain

cyclophosphamide

Sciences

de la vie / Life Sciences

the

experimental

paradigms

of which

were

were injected first with 1 mL saline and second or in a series of four fractions at hourly intervals. animals having survived for 4 h after the initial

D. Menbtrey

et al. of the ---a--

1250

1

CP 100 mgikg + NaCl

C

NaCI+

mesna300

(once)

mg/kg(once)

T

spinal

trigeminal

(STn).

nucleus

This

last

T

also accompanied, at the same rostrocaudal contralateral staining of the most dorsomedial the trigeminal subnucleus caudalis, in its reaches the cuneate nucleus. 3.2. I i5 z .2 c 9"

2501,

2

1250 1

1

I 30

, 50

_ I 70

I 90

I 110

I 130

-.a--

CP 100 mglkg + N&1(4X1/4)

--t

NaCI+

1000

mesna 300 mg/kg(4X1/4)

I 170

I 190

T

,.k

I 210

(,,,.. ’I’

T -1..

,,I ,_,..__!.,...-.k

750

. T __,__. '/..

500 I

.,' I'

.I. ,..+/.Lg

250

I

I, 30

50

P

/--'

*

A

I 70

I 90

I 110

a

=

I 150

I 170

.

Post-initial Figure 1. Mean behavioural groups of animals having (300 mg/kg total dose, full

I 130

a

injection

I 210

I 230

.

time

response curves during a 4-h period for developed cystitis from either mesna lines) or CP (100 mg/kg, dotted lines).

Groups

Saline + saline CP + mesna

Doses

of mesna (mg/kg). Single injection

40 100 200 300

CP + saline All injections were of 1 mL volume and performed conditions mesna prevented the full development having survived for 4 h after the initial injection.

Mean

a single injection of mesna both behavioural modifications in the range of all the doses injections

of mesna

was

never able to and disease imwe have tested.

had

various

effects

accor-

observed with the paired CP + saline group (3.5 & 0.5) and close to that seen in controls (1.1 f 0.1). The effects on behaviour were also quite impressive although the dose of 200 mg/kg could not prevent transient nor moderate behavioural alterations (figure 3). Mesna at the total dose of

in groups of animals having (CP + mesna) or CP + saline bladder 1.2 2.0 1.8 1.5 2.1 3.8

mg/kg, compared to 40 and modifications were both to the CP + saline group

0.2 (table //I) - thus corresponding to simple chorionic oedema. Bladder-state values, in all of these cases, were significantly (P <: 0.004 to < 0.005) lower than those

(100 mg/kg in 1 mL saline) + NaCl (0.25 mL, triangle) followed by NaCl(3 x 0.25 mL, dots) at hourly intervals (dotted line). In contrastto CP no significant effects on behaviour were obtained, even if mesna at this dose of 300 mg/kg could generate mild cystitis (table 0.

diseases + mesna

CP in

injected dose and were all significantly different from that observed in the paired CP + saline group (3.8 + 0.1; table /I). The bladder states of animals having been uroprotected by a single dose of mesna consequently consisted of a mean of only a simple chorionic oedema development. The best effects on inflammation were obtained at

Multiple

were injected by fractions as follows: either 1 mL saline + mesna (75 mg/kg in 0.25 mL saline, triangle) followed by mesna (3 x 75 mg/kg in 0.25 mL each, dots) at hourly intervals (full line) or CP

(+ SE) of histopathological bladder cyclophosphamide (CP, 100 mg/kg)

reduced a reduction

ding to the total dose of injected drug. Good uroprotection was observed at doses of 40-200 mg/kg. Mean bladder states in these cases were graded from 1.3 f 0.3 to 1.6 f

B. Animals

II. Mean values (saline + saline),

of mesna

bladder disease and a decrease in associated behavioural impairments. Mean values of bladder disease in these cases ranged from 1.5 f 0.5 to 2.1 f 0.1 depending on the

effect, abolish pairment

paradigms were similar to those used when studying the uroprotective effects of mesna on CP cystitis. A. Animals received two injections at same time of 1 mL volume each /;zzngle), either saline (NaCI) + mesna (full line) or CP + NaCl (dotted

only

levels, by a portion of portion that

(figure 2). The best effects on behaviour were observed at doses ranging from 100 to 300 mg/kg. A dose of 40 mg/kg was less effective although bladder disease was reduced. Thus, and in spite of an unquestionable uroprotective

(mins)

Behavioural scores (expressed as mean f SE) are shown on the ordinate, the basal behavioural score being 300. An entirely abnormal behaviour score would be 1 500. Post-injection times, expressed in minutes, are shown on the abscissa. As for table 1, the experimental

Table

effects

doses ranging from 100 to 200 300 mg/kg (table /I). Behavioural delayed and lowered compared

-/

I 190

Uroprotective

Single injections of mesna markedly cystitis-related manifestations with both

, 230

T .d ....J

T

B

i

I 150

subgroup

of staining corresponds to the transitional zone between subnuclei caudalis and interpolaris, thus comprising the ventral paratrigeminal nucleus (vPaT). This staining was

state

been injected (CP + saline).

Fischer’s PLSD saline + saline

* 0.2 + 0.2 f 0.8 * 0.5 kO.1 f 0.1

intraperitoneally

versus group

P= P= P= P=

NS NS

P= 0.01

at the same time. Mesna was injected at doses of 40, 100, of CP cystitis and with the best effects at doses of 100 and 200

Sci.

Paris,

either

saline

Fischer’s PLSD versus CP + saline group

P = 0.02

C. R. Acad.

with

0.000 0.000 0.000 0.000

4 4 2 4

200 and 300 mg/kg. Under these m&g. The data are from animals

Sciences

de

la vie / Life Sciences 1999,322,505-515

Mesna,

----.---.

CPIOO q/kg+

A

CP +mesna4Omgikg

300 mg/kg completely. graded 2.6

NaCl(once) (once)

T

was unable The mean + 0.3 (table

cyclophosphamide

and

cystitis

to prevent cystitis development bladder state in this case was I//) - thus intermediate between

those of paired sham (1 .l f 0.1, P = 0.000 2) and CP + saline (3.5 + 0.5, P < 0.05) groups. This value was also significantly higher than that observed with animals having received CP + mesna in a similar way but at lower doses (table I//). Behavioural modifications that CP-cystitis normally induced such a high dose *

1250

0

30

50

70

90

110

130

...-..-..

CP IM) mgikg + NaCl (once)

--

CP+mesnalOOmg/kg((once)

B

1000

150

170

190

T _,.._L--

,:’

[J;&JT ?jJJ 90 3 b '5 2

110

130

150

170

190

210

230

A

1250-.

..-.----

CP 100 me/kg + NaCl(o"ce)

--c

CP+ mesna200mgikg(once)

T

2 ,,._,L.-¤

750I 500-

250-1

, 30

i!

1250

, 50

, 70

, 90

, 110

, 130

..-.....-

CP 100 mg/kg + NaCl (once)

---e

CP+mesna300mg/kg(once)

1000

I 150

, 170

190

I 210

, 230

j .._.__I/.'.

D

Spinal

230

animals saline mesna

! _____.p"'

1

210

,j’ T

750

and

were (figure

brain

not distinctly 3).

activities

were

affected studied

by mesna

at

in CP-injected

having been (CP + mesna groups) or not (CP + groups) protected from cystitis development by administration. Mesna-protected animals were se-

lected on the basis of their presenting no bladder injury nor behavioural impairment, as could be judged from bladder histology and behavioural observations. As stated above, these uroprotected animals belonged to those which received mesna at low total doses (40 or 100 mg/kg) and by fractions at hourly intervals. were compared to those of saline shams (animals been repetitively shams (animals mesna at doses same experimental in each group.

Results having

injected with saline only) or mesna having been injected with saline and of 40 or 100 mg/kg) and following the protocol. Four animals were included Closer attention was given to the sacral

spinal cord, dorsal paratrigeminal dorsal vagal complex (DVC) which, correspond to the most representative

nucleus (dPaT) and as previously shown, viscero(noci)cep-

tive structures. Compared to basal Fos expression, as gauged in uninjected control animals, sham animals, repetitively injected with either saline or saline + mesna, showed an increase in their Fos activity at both sacral spinal and DVC levels (table IV). The effects were particularly strong for the superficial layers of the dorsal horn in case of saline + mesna injections (m = 18 f 3, P< 0.000 1). The dPaT activity was not modified. CP injection, generating cystitis, resulted in a significant further increase in Fos activity in the sacral parasympathetic column (SPN, m = 16 f 4, P= 0.001). This increase was concomitant to those of the dPaT (m = 31 + 11, PC 0.004) and DVC (m = 157 f 50, P< 0.02). Surprisingly, lamina 1 Fos activity in these tended to decrease. The control of cystitis by mesna had minor effects on Fos staining in all these structures. Thus, although the increase in SPN was reduced (m = 10 f 3, P= 0.05) but not stopped, Fos activities in conditions

,J..’

500 ,,p. _...’ a---m

250, 0

.

--5

I 30

a 50

=

=

e

2

=

I 70

I, 90

110

I 130

I 150

I,, 170

190

210

I 230

other

Post-initial injection time (mins)

injected at increasing 200 mg/kg; 2D: 300 just after CP (triangle). legend of table II. The of 100 to 300 mg/kg abnormal behaviours

doses (2A: 40 mg/kg; 28: 100 mg/kg; 2C: mg/kg). Mesna and saline were injected once See other details for experimental paradigms in graph details are as in figure I. Doses of mesna increased the delay of onset and reduced the more successfully than with a 40 mg/kg dose.

CR. Acad. Sci. Paris, 1999,322,505-515

Sciences

de la vie

/ Life Sciences

were

not

affected.

4. Discussion

Figure

2. Mean behavioural response curves during a 4-h period for groups of animals having been injected intraperitoneally with either CP + saline (dotted lines) or CP + mesna (full lines). Mesna was

areas

tive

The present effects of

study, mesna

which has confirmed the uroprotecon CP cystitis [I O-l 31, has proved

that these effects depend on the dose and on the way the drug is injected. Of all the combinations we have tested only the doses of 40 and 100 mg/kg/i.p, when injected by fractions to compensate the short clearance time of the drug (1.4 h) as compared to that of CP, were the most efficient in controlling CP-cystitis development and its

511

D. Men&rey

et al

Table III. Mean values (+-SE) of histopathological only (saline + saline), cyclophosphamide (100 Groups

bladder diseases in groups mg/kg) + mesna (CP + mesna)

Doses of mesna (mg/kg). Fractioned injections

Saline + saline CP + mesna

Mean

40 100 200 300

CP + saline Following

an initial

starting the full

just after the development

injection

of either

initial injection. of CP cystitis

saline

or CP, saline

Mesna was injected except at the highest

or mesna

bladder

1.1 1.6 1.6 1.3 2.6

fO.l + 0.2 f 0.2 * 0.3 f 0.3

3.5

f 0.5

was

at total doses dose. The data

associated behavioural manifestations. In contrast, single injections, resulting in a mesna release which did not entirely cover the clearance time of CP, resulted in the appearance of late and mild cystitic effects. Mesna at the total dose of 300 mg/kg proved to be the upper limit to protect bladder integrity against CP urotoxic actions, especially when the drug was injected by fractions at hourly intervals. The signs of cystitis observed in this case mimicked, although at a lesser degree, those produced by CP when injected with saline. Cystitic manifestations under these conditions would result from the late release of mesna, which itself has mild bladder toxicity at high doses (present study), and at time points during which the amount of acrolein, the main toxic CP metabolite with which it complexes, has dropped in urine. The acrolein excretion peaks 30 min after CP injection and returns close to normal 90 min later [I 81, forcing the mesna injected after this time to be excreted when urine is almost free of acrolein, thus preventing complexing. In this case, unbound mesna would be toxic enough to possibly wea-

of animals having been injected or CP + saline (CP + saline). state

Fischer’s PLSD saline + saline

Mean

injected

by four

Controls Saline + saline. Fractioned injections Saline + mesna (40/l 00 mg/kg). Fractioned injections CP + saline. Fractioned injections CP + mesna (40/l 00 mg/kg). Fractioned injections

1 f0

3.4

P<

intervals,

0.05

the first injection

Under these conditions for 4 h after the initial

of the series

mesna prevented injection.

with the release of residual CP

I

cord (Lamina 1, dorsal gray commissure [DCC], sacral complex (DVC) in groups of animals having received of 40 or 100 mg/kg (third line), cyclophosphamide (CR

cord

dPaT

DVC

DGC

SPN

3fl 8+2

1 *o 4fl

3+1 3+2

63k20

6~k2

4+2

47+

31 fll 35f14

157+50 140*37

1*0

18&3

7+1

* 0.4 1 f0

9f2 12fl

8+3 lOf2

The data from uninjected animals (controls) are shown of cystitis except for animals injected with CP + saline Four animals for each group, only those injected with parasympathetic activity (SPN column) was reduced activity (dPaT and DVC columns) was not prevented

at hourly mg/kg. survived

0.005 0.005

Repetitive intraperitoneal injections of mesna induced Fos activities in superficial layers of the dorsal horn and at the brainstem level, in a reticular/trigeminal area made up of both the ventrocaudal reticular formation of medulla (vcBRF) and the adjacent ventral paratrigeminal nucleus (vPaT). Reticular/trigeminal activity was predominantly contralateral. The ventral medulla oblongata is an area involved in generatingvasomotor sympathetic and cardiovagal tones (references in [I 91) and known to express Fos in response to various nociceptive and stressful1 inputs (references in [6]) while the vPaT, a trigeminal subarea, is where cephalic inputs of cornea1 [20-281, nasal [29-311, facial skin [27, 281, lingual [27, 321, temporomandibular [33] and meningeal [34] origins converge. In fact major inputs to the vPaT would originate from the cornea or snout [27]. Another area of staining was the dorsomedial portion of subnucleus caudalis that receives dense projec-

Spinal

2+1 7*4

fractions and 300 having

saline

P < 0.004

P = 0.0002

of 40, 100, 200 are from animals

either

Fischer’s PLSD versus CP + saline group

P< P<

ken the mucous membrane toxic byproducts.

bladder state Lamina

versus group

with

NS NS NS

Table IV. Mean values (+ SE) of bladder states and of number of Fos-positive cells in spinal parasympathetic column [SPN]), dorsal paratrigeminal nucleus (dPaT) and dorsal vagal repetitive injections of saline only (saline + saline, second line), saline + mesna at doses 100 mg/kg) + saline (fourth line) or CP + mesna (fifth line, same doses as on line 3). Groups

intraperitoneally

16f4 10+3

15f4

11

on the first line (from reference [7]). None of these animals showed evident histological signs (first column). Mesna was injected by fractions according to the schedule shown in figure 3. CP (fourth and fifth lines) displayed consistent vagal activity (dPaT and DVC columns). Sacral when cystitis was controlled by mesna (fifth line as compared to fourth). In contrast vagal by mesna (fourth and fifth lines).

C. R. Acad.

Sci.

Paris,

Sciences

de

la vie / Life Sciences 1999,322,50&515

Mesna,

.-.....’

CP 103

mglkg

+ NaCl

(4X1,4)

+

CP 100 ma/ks

+ mema

40 mgkg

(4x114)

12504

T

A IWO-

750-

500-

_/250

_

,

4

30

:

T

y

:

;

-

-

l

50

70

90

110

130

150

170

130

.

12504

.

.

*-

210

230

.

...-.--.

CP 100 @kg

--C

CP 100 mgikg + mesna 100 mgnng (4X114)

+ NaCl (4X114)

J

1000 B

90

9 3 x

(g

1250

c 2

110

-.-.-.-

CP 100 m#kg + N&I (4X1,4)

-f-

CP 100 mslks + mm

500 250

200 Wg

170

190

210

I

T.._, =.. T__. ./” 1

i

_ ;

y

:

70

90

110

0

‘-.....I

T T

A

,_..’ _ __... -’ , , 30 50

, 2

‘. T

,l”

2 d ,I,_.... I..;.,”

230

(4X114)

T

.

125oj

150

I . ..’ ..’ -..

5s 750C 1003

130

i( 130

, 150

. 170

.

130

210

230

.

-.-e----

CP 100 mg/kg + NaCl (4X114)

+

CP lWm(ykg+merns3Wm*(4X1N)

_

IWO-

7M-

500-

250

, 2

, 30

r

, 50

, 70

, 30

, 110

Post-irYitial inject& Figure

3. Mean

behavioural

response

I 130

, 150

170

190

210

230

time (mYna) curves

during

a 4-h

period

for

groups of animals having been injected intraperitoneally with either CP + saline (dotted lines) or CP + mesna (full lines). Triangle and dots on abscissa show the injection times. Mesna and saline were injected in four equal fractions with the first injection (first dot) at the same time as that of the CP (triangle) and the succeeding ones every hour (three last dots). See other details for experimental paradigms in legend of table 111. The graph details are as in figure 2. Mesna was injected at increasing doses (3A: 40 mg/kg; 38: 100 mg/kg; 100 m&g) CP; however behavioural

3C: 200 mg/kg; 3D: 300 mg/kg). Mesna stopped (40 and or reduced (200 mg/kg) abnormal behaviours induced by 300 mg/kg of mesna associated with CP produced impairment.

CR. Acad. Sci. Paris, 1999.322,505-515

Sciences

de

la vie

/ Life Sciences

cyclophosphamide

and

cystitis

tions from the tongue and the lower lip [271. The contralateral asymmetry of these activities proved their local origin and seem to result from the intraperitoneal injections, the nociceptive impact of which has been demonstrated [35]. However, the exact nature of these activities cannot be specified since intraperitoneal injections involve both somatic (skin, somatic peritoneum) and visceral (visceral peritoneum) tissues. The demonstration of non-cephalic inputs to these ‘trigeminal’ subareas was quite surprising. A working hypothesis is that the convergent reticular/trigeminal subregion underlined in this study could be part of the anatomical network where pain-related reflexes of various origins are elaborated. Referring to cystitis these reflexes would comprise facial mimics, cephalic reflexes (ocular discharge, pupillary movements), negative cardiac inotropism (bradycardia and hypotension 1361, bradypnea [371) and bladder tone modifications leading to dysuria 1381. Regarding the bladder tone, it can be mentioned that the ventrolateral reticular formation would act in the same way as the pontine micturition centre to induce bladder contraction, thus, opposite to the DVC which causes bladder relaxation 1391. Contralateral reticular/trigeminal activity could not be detected in the presence of CP, the injection of which generates vagal inputs. This observation could be related to the fact that vagal inputs are known to act on nociceptive reflex and are able to generate antinociception [40, 411. It was disappointing to observe that animals protected from CP-cystitis development by mesna, i.e. those for which both bladder impairment and behavioural alterations could not be observed, only showed minor differences in their Fos activities compared to those of animals having developed the disease. Activities were reduced in SPN but not abolished while others, including dPaT and DVC activities, remained unchanged. Spinal activity other than that of the SPN seems to be of peritoneal origin as it increased with repetitive injections of saline and/or mesna. The dPaT activity, which is largely of vagal origin [7], would originate from the systemic actions that the CP can have when circulating in the blood. For example, CP has been shown to induce nausea and learned taste aversions [42] -this latter resulting in vagal activity [43]. These CP-systemic actions cannot be prevented by mesna as this chemical does not act on CP itself nor on its hepatic metabolites but only counteracts the release of acrolein at the renal level [l O-l 31. Vagal activities could also originate from the bladder itself, if indeed as Jancso and Maggi I441 claimed, some direct bladder afferents join the vagal nerve. In conclusion, it has been confirmed that mesna can effectively protect against CP-cystitis. The uroprotective effect of mesna, however, requires appropriate posologies. This uroprotective effect has led to the confirmation of the spinal actions of the disease, probably via the pelvic nerve, but did not allow a clear distinction between the consequences of the systemic (vagal) and local (spinal, pelvic) effects of CP at supraspinal level.

513

D. Men&rev

et al

Acknowledgments: The authors are indebted to P. Sanderson for her kindness R. Rambur for preparing the illustrations. This study was supported by funds (Inserm) and the Association pour la recherche sur le cancer (Arc, no 9316). scientifique

in preparing the English from the lnstitut national D. Menetrey is supported

text. They are grateful to A. Men&my and de la recherche medicale et de la Sante by the Centre national de la recherche

(CNRS).

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/ Life Sciences

receiving

1431 Houpt T.A., Philopena Increased c-fos expression conditioned taste aversion 1-5.

cyclophosphamide

and

cystitis

I.M., Wessel T.C., Joh T.H., Smith C.P., in nucleus of the solitary tract correlated with to sucrose in rats, Neurosci. Lett. 172 (1994)

[441 Jan& G., Maggi C.A., Distribution bladder afferents in the rat spinal cord,

Brain

of capsaicin-sensitive Res. 418 (1987)

urinary 371-376.