Experimental ocular toxocariasis - British Journal of Ophthalmology · Murine ocular toxocariasis...

British Journal ofOphthalmology, 1984, 68, 89-96

Experimental ocular toxocariasis: a mouse modelS. Y. A. GHAFOOR,' H. V. SMITH,2 W. R. LEE,' R. QUINN,3AND R. W. A. GIRDWOOD2

From the i Tennent Institute ofOphthalmology, Western Infirmary, Glasgow;the 2Bacteriology Department, Stobhill Hospital, Glasgow; and the 3Zoology Department,University of Glasgow, Glasgow

SUMMARY Male mice, strain C57 black, were infected with Toxocara canis by a single intragastricdose of 1500 infective eggs. The eyes were studied at sequential time periods after infection (6 to 63days) by conventional microscopic techniques, and the histological characteristics of the inflam-matory response were recorded. In the majority of animals the disease was unilateral. Twenty-sixlarvae were found in the retina, in the retinal vessels, and in the subretinal space in 20 eyes, while in29 eyes there were inflammatory changes which were not related to the presence of intact orfragmented larval forms. The inflammatory reaction began as a polymorphonuclear response butafter day 13 became a granulomatous reaction. This suggests that the inflammatory phenomenonmay be propagated by the secreted surface antigens in the absence of the living or dead larvae.

Children and adults'2 become infected by theaccidental ingestion of dog faecal material containingToxocara canis ova. The clinical manifestations ofocular toxocariasis are solitary and usually occur inolder children (average 7-5 years) without thegeneralised eosinophilia, hyperglobulinaemia, andhepatomegaly characteristic of visceral larva migrans(VLM).3 Lesions occur unilaterally as a solitaryretinal granuloma in the posterior pole in proximity tothe disc and macula and/or as chronic endophthalmitiswith retinal detachment.4 Wilder- found nematodelarvae in 24 cases of pseudogliomata, and Nichols6identified these larvae as Toxocara canis. In suchstudies eyes were usually enucleated because of thedifficulty of excluding retinoblastoma.

Although ocular toxocariasis is an uncommon formof uveitis and retinitis in the routine uveitis clinic,7treatment of the majority of cases in which there is anexudative retinal detachment is unrewarding. Subtotalpars plana vitrectomy8 has been added recently to thecurrent therapeutic measures, which included topicaland systemic corticosteroids, anthelminthic therapy,and laser photocoagulation. The pathogenicmechanisms which lead to retinal damage are poorlyunderstood, notably the immunopathological inter-reactions between the host and the parasite, so thatthere is a dearth of information on which to base

Correspondence to S. Y. A. Ghafoor, FRCS, Tennent Institute ofOphthalmology, Western Infirmary, Glasgow G1l 6NT.

therapy. It is therefore of value to develop cheap andreliable animal models with a low degree of mortalityand a high degree of morbidity in order to apply therapidly developing techniques of immunohisto-chemistry to the study of nematode-induced inflam-matory disease. To this end we present our findings ina preliminary study of ocular toxocariasis in themouse. To our knowledge there has not been adetailed histological study over an extended period ofthe effects of toxocariasis on the pigmented eye of alaboratory animal.

Materials and methods

Animals. Three uninfected 10-week-old mice (C57black strain) were used as controls to determine thenormal ocular histology in this strain. Twenty-sevenmale C57 black mice, 10 weeks old, were infectedwith a single dose of 1500 infective eggs by intragastricintubation on day 0, and 3 animals were chosenrandomly and killed by cervical dislocation underanaesthesia on days 6, 13, 20, 32, 34, 41, 50, 56, and 63after infection.

Isolation ofinfective eggs. Toxocara canis eggs wererecovered from the uteri of adult female wormsobtained from the gastrointestinal tracts of dogs atautopsy. The worms were washed and stored in 4%formalin at room temperature (21°C) for 4 weeks.Following embryonation, which occurred while theadult worm was immersed in formalin, and develop-

. 89

on July 12, 2020 by guest. Protected by copyright.

http://bjo.bmj.com

/B

r J Ophthalm

ol: first published as 10.1136/bjo.68.2.89 on 1 February 1984. D

ownloaded from

http://bjo.bmj.com/

S. Y. A. Ghafoor, H. V. Smith, W. R. Lee, R. Quinn, and R. W. A. Girdwood

Fig. 1 Illustrations ofToxocara canis larva in the retina (paraffin sections stainedHand E). a: Toxocara canis larva in lumenofretinal capillary atday 13 (T) (x630). b: Normal retina ofa control mouse (Hand E, x400). c: Toxocara canis larva lyingin superficial retina; the adjacent vessel ( a ) shows a vasculitis (x400). d: Larva within a retinal capillary and the vitreous atday 56 (x630). e: Larva in outer segments at day 6 (x630). f: Larva within a subretinal haemorrhage at day 34 (x630).

ment to the second larval stage (L2) within theeggshell, the infective eggs were dissected from theuteri and washed extensively in phosphate bufferedsaline (PBS) pH 741, prior to use.

Pathological examination. The eyes and orbitaltissues were removed immediately after death andwere fixed in 2-5% gluteraldehyde in 0-4 Mcacodylate buffer, pH 7-2, at 4°C. Each eye wasopened and the lens was removed. The retina wasexamined for lesions under a dissecting microscope atappropriate magnification.

Histological examination. Specimens for lightmicroscopy were embedded in paraffin, and serialsections 5 ,um in thickness were taken in the sagittalplane of the eye and stained with haematoxylin andeosin. Selected sections were stained with carbolchromotrope to demonstrate eosinophils. To assess

the viability of L2 Toxocara canis larvae one samplefrom a bisected eye from each group of 3 mice wasretained for transmission electron microscopy(TEM). These samples were rinsed in cacodylatebuffer (0-2 M), postfixed in osmium tetroxide (2%),dehydrated in graded alcohols, and embedded inAraldite. Semithin sections were taken and stainedwith toluidine blue. For the study of the preservationof the larval forms ultrathin sections were stainedwith uranyl acetate and lead citrate and examined in aPhillips 301 electron microscope.

Results

Macroscopic and microscopic examination of thecontrol material revealed the normal architecture ofthe pigmented mouse eye (Fig. lb).

90

Aj 71FI,


http://bjo.bmj.com

/B

r J Ophthalm


ownloaded from

http://bjo.bmj.com/

Experimental ocular toxocariasis: a mouse model

Larval Incidence

R

Mouse

R

Mouse 11

R

Mouse

0 0~~0

* 0 0 0 0 0

* 00 0 00 0 0

0 6 13 20 32 34 41 50 56 63

DAYS KILLED

Fig. 2 Larval incidence in eyes ofinfected mice. R is theright eye, L is the left eye. I, HI, and III denote a set of3randomly chosen mice killed on the days noted in thehorizontal axis.

Macroscopic examination of the 54 eyes from 27animals, which had been infected with Toxocaracanis larvae revealed grey fluffy masses in theperipheral retina in 5 eyes only. Twenty-six larvaewere found in serial sections from the experimentaleyes (Table 1) and were recognised at each timeinterval throughout the experimental period (Fig. 2).The location of the larvae was mainly at the posterior

Table 1 Distribution of larvae in 54 eyes

Distribution of larvae

Posterior pole 18Peripheral retina 5Vitreous 1Retinal capillary 2Total 26

pole (Table 1), but 5 were in the peripheral retina and

one was present in the vitreous. On 2 occasions larvae

were identified within the lumen of retinal capillaries

(Fig. la', d), while the majority appeared to be lyingwithin the retinal substance or were in the subretinal

space (Fig. le, f).The pathological changes which occurred in the

eyes of the infected animals are summarised in Table

2. Evidence of occlusive retinal disease was obtained

most frequently in the early stages of infection (days 6

and 13), when superficial and deep haemorrhageswere observed; the latter extended in some cases into

the subretinal space (Fig. if). Microinfarcts were

found commonly throughout the experimental period(Fig. 3a, Table 2). Haemorrhagic pseudocysts were

found in the retina and the vitreous (Fig. 3b). Retinal

vasculitis (Fig. lc) was more frequently found in the

later stages of the experiment.It is noteworthy that on only one occasion was

there evidence of an inflammatory reaction in the

presence of a larval form (Fig. 3), and it was rarely

possible to identify structures which could have been

interpreted as dead or necrotic larval forms.

Ultrastructural examination of the larvae showed the

organism to have normal cell walls and organelles

(Fig. 4).

Nongranulomatous inflammatory infiltrates, con-

sisting of lymphocytes and plasma cells were found in

the anterior uvea and the pars plana (Fig. 3d, e). This

type of cellular infiltrate succeeded a polymorpho-nuclear leucocyte infiltrate which occurred in the first

13 days. The vitreous was commonly involved in the

inflammatory process, but the infiltrates were mainly

lymphocytic and haemoffhagic, and the macrophageswithin the vitreous body contained melanin and red

cell degradation products (Fig. 3d, e, f). One circum-

scribed abscess was found in the vitreous adjacent to

an intact larva (Fig. 3c).A granulomatous response was rare and, apart

Table 2 The incidence of histopathologicalfeatures in experimental toxocariasis. The numbers in each column refer to the

number ofeyes affected: 6 eyes were examined at each specified time period

Lesion Day 6 13 20 32 34 41 50 56 63 Total

Iridocyclitis 0 0 5 5 1 2 3 1 3 20Pars planitis 0 2 6 4 3 1 5 2 6 29Vitreal haemorrhage 3 3 2 1 4 4 1 1 9 28Vitreal infiltrate 3 2 5 4 4 4 6 3 6 37Optic nerve papillitis 0 0 1 1 0 0 1 2 2 7Retinal superficial haemorrhage 4 0 1 1 1 0 1 1 0 9Retinal deep haemorrhage 3 0 0 1 0 1 1 0 0 6Subretinal haemorrhage 4 1 1 1 0 1 0 0 0 8Microinfarcts 3 4 1 4 3 3 3 4 2 27Vasculitis 1 3 1 3 1 2 2 1 1 15Subretinal infiltrate 0 0 0 4 1 2 2 2 0 1 1Retinalgranuloma 0 3 3 0 0 3 1 0 0 10Choroid 0 0 0 0 0 0 0 0 0 0Orbit 0 0 0 0 1 0 0 0 0 1

91

L

L


http://bjo.bmj.com

/B

r J Ophthalm


ownloaded from

http://bjo.bmj.com/

92 S. Y. A. Ghafoor, H. V. Smith, W. R. Lee, R. Quinn, and R. W. A. Girdwood

a b

'e~ ~~~~~~~~~~~~~~~~~~~4f

Li~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~i

**^**V*) 0 ',0+i*B44W*

&I~~~~~~~~~~~~~

Fig. 3 Illustrations ofthe intraocular reactions (paraffin sections stained H and E). a: Superficial microinfarct ( T) at day 6(x400). b: Haemorrhagicpseudocyst (p) adjacentto a retinalgranuloma ( T ) (x 160). c: A mixed inflammatory infiltrate withan intact larva ( t ) in the peripheral vitreous (x400). d: Marked iridocyclitis at day32 (x630). e: Low-grade iridocyclitisandpars planitis with vitreous infiltration at day 41 (x400). f: Macrophages ( red cells, and lymphocytes in the vitreous(x630).


http://bjo.bmj.com

/B

r J Ophthalm


ownloaded from

http://bjo.bmj.com/


r,

^_ ~~~a

Fig.!4 _Electron intactstructur.Fig. 4 Electron micrographs showing intact internal structure

from a solitary lesion in the orbit, occurred in theretina in 10 eyes which had been removed after the6th day (Fig. 3b, 5). The granulomas consisted ofclusters of epithelioid macrophages with small in-conspicuous multinucleate giant cells (Fig. 6a).The clusters of macrophages were surrounded by amantle of lymphocytes and polymorphonuclearleucocytes of neutrophil type. Eosinophil poly-

Retinal Granuloma Incidence

R

Mouse T

R

Mouse 11

L

R

Mouse IFI

0 6 t3 20 32 34 41 50 56 63

DAYS KILLED

Fig. 5 Retinal granuloma incidence in eyes ofinfectedmice. R ifthe righteye, L is the left eye. I, 11, and III denote aset of3 randomly chosen mice killed on the days noted in thehorizontal axis.

A..

. _ _ b~~~~~~~~

es ofToxocara canis second-stage larva (x2083).

morphonuclear leucocytes were not observed at anystage of the inflammatory process. It was alsoremarkable that the choroid and choriocapillariswere free from inflammatory cell infiltration in all thespecimens examined. Careful search in thesegranulomas failed except on one occasion (Fig. 3c) todemonstrate intact larvae or fragments of larvae.Optic nerve involvement (Fig. 6b) was relativelyuncommon (Fig. 6a), and an orbital granuloma wasfound in only one specimen (Fig. 6c).

Additional features of interest included preretinalcellular infiltration (Fig. 6d) and the presence ofisolated aggregates of macrophages within the photo-receptor layer (Fig. 6e, f).

Discussion

Murine ocular toxocariasis in the C57 black strainoffered a good animal model for study. Previouswork by Olsen' on Swiss Yale mice extended over 34days but did not illustrate the histopathologicalchanges described in the text. Studies on a guinea-pigmodel for ocular toxocariasis considered the role ofIgE antibodies and mast cells in detail,'0 but thisspecies is atypical in its immune response. In additionthis may not be pertinent to the human disease, sincethe guinea-pig does not play a part in the life cycle ofthe parasite, whereas the mouse can be a paratenichost and hence does transmit the disease wheningested by the dog. The present mouse model,

93

L

L


http://bjo.bmj.com

/B

r J Ophthalm


ownloaded from

http://bjo.bmj.com/

94 S. Y. A. Ghafoor, H. V. Smith, W. R. Lee, R. Quinn, and R. W. A. Girdwood

a b

.

w I.xit.

..

N ~ o. Aw,

:,-#t#w._;=i W 4:

SO

001| B!S.2/1f '

w . . .~~~~~~~~~~~~0

Ofl;,:/i

.40.:.

&

E - _~~~~~~~~~~4.1R__ IRF" z4 ..A

4

Fig. 6 Features ofinflammatory process (paraffin sections stained H and E). a: Giant-cell granulomatous reaction in the

retina atday 41. b: Papillitis with vasculitis in central vessels at day 32 (x 630). c: A well circumscribedgranulomatous reaction

external to sclera (S) at day 34 (x630). d: Peripheral retina showing preretinal cellular proliferation ( t ) (x630).

e and f: Clusters ofmacrophages within the photoreceptor layers (x 750) (x630).

3,.W%.1~..9.$

11%.

ivIFWW

S


http://bjo.bmj.com

/B

r J Ophthalm


ownloaded from

http://bjo.bmj.com/


studied over a more extended period, simulated thebasic pathological changes described in humanhistological studies, namely, (a) diffuse nematodeendophthalmitis, (b) posterior pole granulomata,and (c) peripheral retinitis. '

I 11At the cellular level the early responses at day 6

were predominantly those of a neutrophil poly-morphonuclear leucocytic infiltration with vasculitisand retinal microinfarction. From day 13 onwardsthere was an increase in the macrophage component,and the distribution of these infiltrates suggested thatthe sites of entry were mainly the pars plana, the opticdisc, and the retinal (but never the choroidal) bloodvessels. Although macrophages were found withinthe outer segments in the subretinal space, and ingeneral there was diffuse retinal infiltration, thechoroidal stroma was notably free from infiltration.Marked iridocyclitis and pars planitis were found.

Hypopyon or iris nodules were not observed on

external examination as has been reported in humancases. 12Granuloma formation was noted between days 13

and 50 and occurred mainly at the posterior pole.Although on 2 occasions larvae and granulomas wereseen at separate locations within the same eye, on

only 2 occasions was a larva or the remnants of a larvaseen within a granuloma in the many serial sectionswhich were examined. The granulomatous reactionconsisted of macrophages, multinucleate giant cells,lymphocytes, and plasma cells, but eosinophils wereinconspicuous even with the application of a specialstain. The possibility of degranulation of eosinophilscannot, however, be excluded as an explanation fortheir apparent absence from the tissue. It is relevantto add that, in the myotropic phases of murine toxo-cariasis, eosinophils are present in the early poorlydefined granulomas but are rapidly replaced by epi-thelioid macrophages in the mature granuloma in thethird week of infection. '3 By contrast in the neuraltissue in the present model'4 and unpublished data(Smith) there is little or no response around migratingsecond-stage larvae or their migratory pathways.Thus the inflammatory changes seen in the oculardisease may represent an intermediary response inthe myotropic and neurotropic phases due to theunique character of the formative tissues of the eye.The route of entry of larvae into the eye is vascular,

and in this study larvae were demonstrated in retinalcapillaries. None was found in the choroidal vessels.This is pertinent to the clinical observation'" whichhas shown that larvae can enter the eye via the retinalblood vessels before entry into the vitreous.Perilimbal infiltrates (composed of polymorpho-nuclear leucocyte cells) were noted, but larvae were

not found in the cornea or-anterior segment, thoughtheir presence has been reported clinically.3

A consistent finding in this study was that larvae orlarval remnants were not seen within the centre of agranuloma. The presence of a larva or larval remnantswithin a necrotising granuloma containing eosinophilsis, however, a diagnostic pathological feature ofhuman toxocariasis.' The apparent absence of larvalforms from the ocular granulomas observed in thismodel implies that the larvae do not have to be presentto propagate the mature granuloma which may havebeen stimulated initially by them. This suggestion issupported by the observation that larvae are not seenin early granulomas in murine visceral larva migrans,and Kayes and Oakes'3 have suggested that Toxocarasecond-stage larvae can migrate out of an earlygranuloma.

Toxocara sp. second-stage larvae are rapid tissuemigrators which have individual tracts in the tissuesthrough which they migrate. Recent work has shownthat the larvae have a capacity for rapid metabolicturnover of their outer antigenic glycosylated surfaceproteins. 16 This turnover is complete within 3 hours,and antibodies raised against the excretions/secretionsare unable to bind to the outer larval surface at bodytemperature. This rapid surface turnover mayaccount for the inability of antibody-producing orcytotoxic immune cells, individually or in combina-tion, to attach to the surfaces. By this evasivemechanism the parasite may avoid larval entrapmentwithin a granuloma. Thus, although the larvae ortheir products may incite granuloma formation, theircontinued presence may not be necessary for thepropagation of the immune response. The hypothesisthat by rapid migration' and surface protein turnoverlarvae escape entrapment would account for the factthat inflammatory cells were inconspicuous in thesurrounding tissues when larvae were observed in theretina or retinal vessels. Another possible explanationfor the latter observations is that larvae can absorbhost surface antigens on to their surfaces or produceparasite-specific antigens which cross-react withblood group antigens,'8 thus providing them with anantigenic disguise. '9

We thank James Ralston and Dorothy Aitken for their valuabletechnical help, Jennifer Murray for typing the manuscript, JohnHendry for the photographs, and the Medical IllustrationsDepartment, Western Infirmary, for the diagrams.

This work was supported in part (H.V.S. and R.W.A.G.) by MRCgrant no. G8118073/T.

References1 Duguid IM. Features of ocular infestation by Toxocara. Br JOphthalmol 1961; 45: 789-96.

2 Dean-Hart JC, Raistrick ER. Adult toxocariasis. TransOphthalmol Soc UK 1977; 97:164-7.

3 Brown DH. Ocular Toxocara canis. J Pediatr Ophthalmol 1970;7: 182-92.

4 Duguid TM. Chronic endophthalmitis due to Toxocara. Br JOphthalmol 1961; 45: 705-17.

95


http://bjo.bmj.com

/B

r J Ophthalm


ownloaded from

http://bjo.bmj.com/

S. Y. A. Ghafoor, H. V. Smith, W. R. Lee, R. Quinn, and R. W. A. Girdwood

5 Wilder HC. Nematode endophthalmitis. Trans Am AcadOphthalmol Otolaryngol 1950; 55: 99-109.

6 Nichols RL. The aetiology of visceral larval migrans. Diagnosticmorphology of infective second stage toxocara larvae. J Parasitol1956; 42: 349-62.

7 Perkins ES. Patterns of uveitis in children. BrJ Ophthalmol 1966;50:169-85.

8 Belmont JB, Irvine A, Benson W, O'Connor R. Vitrectomy inocular toxocariasis. Arch Ophthalmol 1982; 100: 1912-5.

9 Olson U. Ocular toxocariasis in mice. Int J Parasitol 1976; 6:247-51.

10 Rockey JH, Donnelly JJ, Stromberg BE. Immunopathology ofascarid infection of the eye. Arch Ophthalmol 1981; 99:

1831-40.11 Wilkinson CP, Welch RB. Intraocular Toxocara. Am J

Ophthalmol 1971; 71: 921-30.12 Liesegang TJ. Atypical ocular toxocariasis. J Pediatr Ophthalmol

1971; 14: 349-53.

13 Kayes SG, Oakes J. Development of the granulomatous responsein murine toxocariasis. Am J Pathol 1978; 93: 277-94.

14 Abo-Shehada. Studies of some factors affecting the migrationand survival of larval Toxocara canis within two strains of mice.PhD thesis. University of Wales: 1982.

15 Byers B, Kimura SJ. Uveitis after death of a larva in the vitreouscavity. Am J Ophthalmol 1974; 77:63-6.

16 Smith HV, Quinn R, Kusel JR, Girdwood RWA. Effects oftemperature and antimetabolites on antibody binding to outersurface of second stage Toxocara canis larvae. Mol BiochemParasitol 1981; 4: 183-93.

17 Croll NA, Mathews BE. Biology of nematodes. Glasgow:Blackie, 1977: 79.

18 Huntley CC, Lyerly A, Patterson MV. Isohemaglutinins inparasitic infections. JAMA 1969; 208: 1145-8.

19 Ogilvie BM, Worms MJ. Immunology of nematode parasites. In:Cohen S, Sadun EH. eds. Immunology of parasitic infections.Oxford: Blackwell Scientific Publications, 1976: 380-3.

96


http://bjo.bmj.com

/B

r J Ophthalm


ownloaded from

http://bjo.bmj.com/

Experimental ocular toxocariasis - British Journal of Ophthalmology · Murine ocular toxocariasis...

Documents

Transcript of Experimental ocular toxocariasis - British Journal of Ophthalmology · Murine ocular toxocariasis...