niemann-pick disease-like inclusions caused by a...

1022 Reports Investigative OphthalmologyDecember 1976

2-fold molar excess as that of 600-fold molar ex-cess. The percentage of specific binding was cal-culated from the difference between the countsin the absence and in the presence of unlabeledRBP. This difference represented 50 per cent ofthe total counts bound to PE in the absenceof unlabeled RBP, for a PE concentration of 8mg. dry weight per milliliter. Results from twotypical experiments are reproduced in Fig. 1.From analysis of the figure, it is evident thatRBP isolated from the plasma of patients withthe recessive form of RP does not demonstrateany appreciable difference from normal humanRBP in its capacity to interact with the RBP re-ceptor present on the cell membrane of normalbovine PE.

Present results seem to definitely demonstratethat no impairment of RBP function exists inthe most common form of RP. They do not ofcourse rule out the possibility that the RBP re-ceptor on the plasma membrane of PE may beabnormal in this disease.

From the Department of Ophthalmology, Uni-versity of Parma, Italy. This work was supportedby grant 75.01056.04/115/2323 from ConsiglioNazionale delle Ricerche. Submitted for publica-tion July 12, 1976. Reprint requests: G. Maraini,M.D., Department of Ophthalmology, Universityof Parma, 43100 Parma, Italy.

Key words: retinol-binding protein, retinol, pig-ment epithelium, retinitis pigmentosa, vitamin A.

R E F E R E N C E S1. Dowling, J. E., and Wald, C : The biological

action of vitamin A acid, Proc. Natl. Acad.Sci. USA 44: 587, 1960.

2. Futterman, S., Swanson, D., and Kalina, R.:Retinol in retinitis pigmentosa: evidence thatretinol is in normal concentration in serumand the retinol-binding protein complex dis-plays unaltered fluorescence properties, IN-VEST. OPHTHALMOL. 13: 798, 1974.

3. Kanai, M., Raz, A., and Goodman, D. W. S.:Retinol-binding protein: the transport pro-tein for vitamin A in human plasma, J. Clin.Invest. 47: 2025, 1968.

4. Maraini, G., Fadda, G., and Gozzoli, F.:Serum levels of retinol-binding protein indifferent genetic types of retinitis pigmentosa,INVEST. OPHTHALMOL. 14: 236, 1975.

5. Maraini, G.: The vitamin A transporting pro-tein complex in human hereditary pigmentousretinal dystrophy, INVEST. OPHTHALMOL. 13:288, 1974.

6. Heller, J.: Interactions of plasma retinol-binding protein with its receptor, J. Biol.Chem. 250: 3613, 1975.

7. Maraini, G., and Gozzoli, F.: Binding ofretinol to isolated retinal pigment epitheliumin the presence and absence of retinol-bind-ing protein, INVEST. OPHTHALMOL. 14: 785,1975.

8. Heller, J., and Bok, D.: A specific receptor

for retinol-binding protein as detected bybinding of human and bovine retinol-bindingprotein to pigment epithelial cells, Am. J.Ophthalmol. 81: 93, 1976.

9. Peterson, P. A., and Berggard, I.: Isolationand properties of a human retinol-transport-ing protein, J. Biol. Chem. 246: 25, 1971.

10. Vahlquist, A., Nilsson, S. F., and Peterson,P. A.: Isolation of human retinol-bindingprotein by affinity chromatography, Eur. J.Biochem. 20: 160, 1971.

Niemann-Pick disease-like inclusionscaused by a hypocholesteremic agent.MACHIKO SAKURAGAWA.

AY9944, an inhibitor of cholesterol biosynthesis,was injected into albino rats and the ocular tissuewas studied by light and electron microscopy.Abundant lamellar inclusion bodies accumulated invarious cells of the eye, especially in the ganglioncells of the retina and glial cells of the opticnerve. Prolonged administration of this drug re-sulted in degeneration of retinal ganglion cellsand oligodendroglial cells of the optic nerve.Micro-organelles of the inclusion body-laden cellswere otherwise twrmal in their appearance. Theelectron microscojnc appearance of these inclusionbodies and their distribution in the ocular tissuesclosely resembled those of Niemann-Pick disease.

Trans-1,4-bis (2-chlorobenzylaminomethyl) cy-clohexane dihydrochloride (AY9944) is one of themost effective hypocholesteremic agents.1 Suzukiand Zagoren2 have extensively reported electronmicroscopic and biochemical observations on thecentral and peripheral nervous systems of develop-ing rats following the injection of this drug. Also,several other authors have described membranousinclusion bodies which abundantly accumulate inthe affected cells.3- 4

The present experiments have revealed that ad-ministration of AY9944 to albino rats causes con-siderable changes in various cells of the eye andthat the accumulation of the inclusion bodiesstrikingly resembles that in Niemann-Pick disease.The purpose of this communication is to empha-size a possible correlation between the effects ofthis drug and the pathogenesis of certain sphingo-lipidosis.

Materials and methods. Sprague-Dawley strainalbino rats were used in this study. AY9944 wasdissolved in physiologic saline solution (5 mg.per milliliter) and sterilized by ultrafiltration. Adaily dose of 50 mg. per kilogram body weight(about 0.35 mg. for a 2-day-old rat) was injectedinto the peritoneal cavity. Animals were injectedcommencing on the second postnatal day (Group

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Fig. 1. The ganglion cell of the retina of a Group I animal following 12 injections. Irregularelectron-dense inclusion bodies are abundantly present in the cytoplasm. Smooth and roughendoplasmic reticulum are enlarged. (*7,600.) Left inset, Higher magnification of the markedarea, Newly formed inclusion bodies are seen bound by membranes and free in the cyto-plasm. (x22,200.) Right inset, Higher magnification of an inclusion body which showslamellar membranes. (x38,700.)

I) and on the third postnatal week (Group II).Adult rats were similarly injected. The animalswere killed on the next day following injection1, 3, 5, 7, 10, 12, 15, and 21. Some animals werekept for 1 and 3 weeks after five injections toobserve the delayed effect. At least three animalswere examined for each time period. Controlanimals were injected with physiologic salinesolution intraperitoneally at identical time in-tervals.

The eyes and entire optic nerve were fixed ina 4 per cent glutaraldehyde solution in 0.15Mphosphate buffer (pH 7.2) and small pieces ofthe posterior retina, optic nerve, cornea, ciliaryepithelium, and lens were excised. Tissues werepost-fixed in 1 per cent osmium tetroxide in thesame buffer solution for 90 minutes, dehydratedwith graded ethyl alcohols, and then embedded inan epoxy resin. Sections cut 1 fim thick werestained with toluidine blue for light microscopy.Ultrathin sections were stained with uranyl acetateand lead citrate and examined by electron mi-croscopy.

Eyes of several animals were embedded ingelatin after formalin fixation and frozen sectionswere cut 10 jam thick and examined under cross-

polarized light for birefringency. Sections werealso stained for fat and by the periodic acid-Schiff (PAS) method.

Results. Weight gain of the animals treatedsoon after birth became markedly retarded afterfour or five injections, and many animals died. Allanimals in Group I that received more than fiveinjections became alaxic and died within thefollowing 2 weeks. Most animals in Group IIgained weight relatively well and did not showsevere neurologic symptoms.

Retina. Twenty-four hours after the injection ofa single dose, electron-dense as well as hyper-chromatic inclusion bodies began to accumulatein a few ganglion cells and some fine processesof Muller's cells of both Groups I and II. Theearliest inclusion bodies in these cells measuredabout 0.15 to 0.2 pm in diameter and appearedusually in the cytoplasmic matrix unconnectedwith micro-organelles. However, they were even-tually bound by membranes and some of themwere fused within lysosomes.

With the further intoxication, these inclusionbodies began to show lamellar structure, com-prising whorled concentric membranes, having awidth of about 75 A without definite periodicity.



Fig. 2. Frozen sections of the ganglion cells of aGroup I animal following six injections. Ganglioncells (arrows) are positively stained with Sudanblack B (A) and PAS (B). Birefringency is demon-strated in the ganglion and some cells in thebipolar cell layer (C). (A and B, x900; C, x600.)

Fig. 3. The pigment epithelium (PE) of a GroupII animal following 10 injections. Inclusion bodieswhich show a crystalloid structure are abundantlypresent in the epithelial cell. OS, Outer segmentof the photoreceptor. (*8,400.) Inset, Higher mag-nification of the crystalloid structure. (x38,400.)

The membranes were often closely packed to forma configuration like that of myelin. The numbersof the lamellar inclusions and the affected cellsincreased markedly within a short period of time.Following five injections, all ganglion and Miiller'scells, several horizontal and amacrine cells, andsome bipolar cells of the retina were involved.Despite the abundance of the inclusion bodies,the cytoplasm of these cells showed no sign ofedema or degeneration during this stage. Thenumber of lysosomes might have been increasedbut all other micro-organelles were normal inappearance.

Deposition of the inclusion bodies in the retinabecame conspicuous after twelve injections. Thelarger inclusion bodies, measuring about 0.3 to0.8 /im in diameter were distributed diffuselythroughout the cytoplasm (Fig. 1). The inclusionbodies in the ganglion cells of Group I animalsconsisted of densely packed membranes and weresomewhat irregular in shape, whereas those of the

Group II animals were loose and round. Manylamellar bodies formed large agglomerates. In-clusion bodies which were fused in lysosomesshowed a moderately positive histochemical re-action for acid phosphatase. Also, they werestrongly stained with PAS and Sudan black B andshowed brilliant birefringency by cross polarizedmicroscopy (Fig. 2).

The cytoplasm of the ganglion cell began toshow some changes at this stage. Enlargement ofsaccular lumens of smooth and rough endoplasmicreticulum became apparent, but the cells stilllacked any sign of degeneration. When poisonedfurther, however, the ganglion cells becameedematous and degenerated. The severely dam-aged ganglion cells were totally vacuolated. Also,shrunken cells with pyknotic nuclei were presentin the bipolar cell layer. These cells eventuallydisappeared from the retinal tissue.

The myelinoid inclusions in the various cellspersisted even when the ganglion cells had begun


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Fig. 4. The postbulbar zone of the optic nerve of a Group I animal following 12 injections.inset, Light microscopic view of the area. (Toluidine blue stain; x700.) A, Lamellar inclu-sion bodies are abundantly deposited in glial cells. The cytoplasm is otherwise normal.(x7,700.) B, Higher magnification of an inclusion body. (x52,800.) C, Some inclusion bodiesshow a positive reaction for acid phosphatase. (x57,600.)

to degenerate. These cells remained normal ex-cept for a small number of cells in the bipolarcell layer which degenerated at a later stage.Inclusion bodies were absent in the differentiatingphotoreceptor cells. Development of the photo-receptor elements was unaffected by the drug ad-ministration.

Pigment epithelium. Abundant inclusion bodieswere present in the pigment epithelial cells simi-lar to those in the ganglion cells. However, de-generative changes in the pigment epitheliumwere mild (Fig. 3). The inclusion bodies in thepigment epithelium often showed fine reticularmembranes which were arranged in a crystalloidpattern (Fig. 3, insert). At the early stage of theintoxication, a transitional step of transformationof smooth endoplasmic reticulum into lamellarbodies and then crystalloid bodies was evident.The crystalloid inclusion bodies were more fre-quently found in Group II animals. Followingten injections, the inclusion agglomerated intoirregular giant granules.

Optic nerve. Pathologic changes in the opticnerve were considerably variable with the ageof the animals. Animals that received the injec-tion prior to myelination {i.e., on the fifth post-

natal day), showed extremely severe damages,whereas older animals produced considerablymilder changes.

Following a single injection, 3-day-old ratsshowed deposition of the inclusion bodies in theglial cells, especially astrocytes in the postbulbarzone. After three injections, marked swelling wasnoted in many axons in these areas.

Following the twelfth injection, almost all gliacells in the postbulbar zone contained abundantinclusion bodies, many of which were conglom-erated into large masses (Fig. 4, A and B). Theappearance of the inclusion bodies was identicalto that of the ganglion cell of the Group II ani-mals. A positive acid phosphatase activity wasdemonstrated within the lamellar inclusion bodies(Fig. 4, C). Degeneration in the axons becameapparent at this stage. Nodular swelling of theaxon was frequently observed in the postbulbarzone. Swollen and proliferated mitochondria andvarious electron-dense particles were abundantlypacked in the axonal nodules (Fig. 5). The num-ber of nerve fibers was reduced and large spaceswere formed between the axons. On further ad-ministration, affected glia cells began to de-generate.



Fig. 5. The postbulbar zone of the optic nerve ofa Group I animal following 12 injections. Anodularly swollen axon contains various electron-dense inclusion bodies. The lamellar inclusionbodies in the glial cell are shown in the upperleft corner. (xlO,600.) Inset, Higher magnifica-tion of the marked area. (x30,000.)

Other ocular tissues. Inclusion bodies havinga similar structure were abundantly present inthe stromal and endothelial cells of the cornea,epithelium and fibers of the lens, ciliary and irisepithelium, and endothelium, and pericytes ofblood capillaries. The inclusion body-laden cellswere otherwise normal in their cytologic ap-pearance.

Discussion. The present investigation has dem-onstrated that AY9944, a hypocholesteremic drugwhich blocks A7-reductase in the cholesterol bio-synthetic pathway, resulting in accumulation of7-dehydrocholesterol, causes deposition of mem-branous inclusion bodies in various cells of theeye, especially in the ganglion cells of the retinaand glia cells of the optic nerve. Similar changesin the brain and optic nerve have been notedby several investigators,2- 5 but the main mor-

phologic emphasis has been concentrated on de-generation of oligodendroglia and retardation ofmyelin formation.

The present study shows that the lamellarinclusion bodies in the AY9944-intoxicated ratsare formed only in the cells, the cytologic struc-ture of which is otherwise normal, and at a stagesignificantly earlier than when the cells begin todegenerate. Although retinal ganglion cells andoligodendroglia of the optic nerve degenerateeventually, many other cells of the eye whichaccumulate the inclusion bodies remain normal.

The fine structural and histochemical appear-ance of the lamellar inclusion bodies in this ex-periment is strikingly similar to those in varioussphingolipidoses. In addition, the wide distributionof the inclusion bodies in the ocular tissue issimilar to that observed in Niemann-Pick diseasereported by Robb and Kuwabara8 and Libert andassociates.7

It has been known that the tissue cholesterol aswell as the sphingomyelin is increased in Nie-mann-Pick disease. Recently Brady8 has sug-gested the presence of a primary disturbance ofcholesterol metabolism in this disease. A pre-liminary biochemical study of the retina and lensof the rats administered AY9944 in this studyhas demonstrated moderate decrease of the tis-sue cholesterol and marked reduction of sphingo-myelinase activity at the early experimental stage.0

Administration of AY9944 seemingly causes in-hibition of the sphingomyelinase in various cellsresulting in the marked deposit of the lamellarinclusion bodies. Further biochemical and mor-phologic studies are now under investigation.

Several authors have suggested a possible originfor the inclusions induced by hypocholesteremicdrugs as an elaboration from the smooth endo-plasmic reticulum.3>10 Also, crystalloid bodieshave been demonstrated in the adrenal cortex ofthe mice treated with AY9944, Triparanol, and20, 25-diazacholesterol.3 This study has demon-strated the continuity of the newly formed ag-gregates of membranes (Chen and Yates, typeII inclusions10) with the smooth endoplasmicreticulum in the pigment epithelium. The ag-gregated membranes form crystalloid inclusionbodies. However, lamellar inclusion bodies inother cells appear to be formed within the cyto-plasmic matrix without any correlation with mi-cro-organelles.

Marked retardation of myelin formation hasbeen demonstrated in the optic nerve of theGroup I rats, as previously reported by Rawlins.5

Also, oligodendroglial degeneration has been con-sistently demonstrated in the optic nerve afterprolonged poisoning. Suzuki and Zagoren2 havepointed out that this oligodendroglial damagecauses the myelin degeneration, leaving axons in-tact. However, the present study has revealed that


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axons in the optic nerve degenerate profoundlyand that the number of axons are markedly re-duced even at the early experimental stage.

The author acknowledges Dr. D. Dvornik,Ayerst Laboratory, for supplying the AY9944 usedin this investigation, and Dr. T. Kuwabara, forhis continuous encouragement.

From the Laboratory of Vision Research, Na-tional Eye Institute, National Institutes of Health,U. S. Department of Health, Education andWelfare, Bethesda, Md. This work was presentedat the Annual Meeting of Association of Researchin Vision and Ophthalmology, Sarasota, Fla.,April, 1976. Submitted for publication July 12,1976. Reprint requests: Machiko Sakuragawa,M.D., Bldg. 6, Rm. 213, Laboratory of VisionResearch, National Eye Institute, National In-stitutes of Health, Bethesda, Md. 20014.Key words: AY9944, retina, optic nerve, electronmicroscopy, hypocholesteremic agents, Niemann-Pick disease, sphingolipidosis, lamellar inclusionbody.

REFERENCES1. Chappel, C, Humber, L., Dubuc, J., et al.:

An inhibitor of cholesterol biosynthesis, Na-ture 201: 497, 1964.

2. Suzuki, K., and Zagoren, J. C : Degenerationof oligodendroglia in the central nervoussystem of rats treated with AY9944 or Tri-paranol, Lab. Invest. 31: 503, 1974.

3. Dietert, S. E., and Scallen, T. J.: An ultra-structural and biochemical study of the ef-fects of three inhibitors of cholesterol bio-synthesis upon murine adrenal gland andtestis: histochemical evidence for a lysosomeresponse, J. Cell Biol. 40: 44, 1969.

4. Zagoren, J. C, Suzuki, K., Bbrnstein, M. B.,et al.: Effect of hypocholesterolemic drugAY9944 on cultured nervous tissue: mor-phologic and biochemical studies, J. Neuro-pathoL Exp. Neurol. 34: 375, 1975.

5. Rawlins, F. A.: A quantitative electron mi-croscopic analysis of myelination in the opticnerve of suckling rats treated with an in-hibitor of cholesterol biosynthesis, Z. Zell-forsch. Mikrosk. Anat. 140: 9, 1973.

6. Robb, R., and Kuwabara, T.: The ocularpathology of type A Niemann-Pick disease,INVEST. OPHTHALMOL. 12: 366, 1973.

7. Libert, J., Toussaint, D., and Guiselings, R.:Ocular findings in Nieman-Pick disease, Am.J. Ophthalmol. 80: 991, 1975.

8. Brady, R. O.: Biochemical approaches to thenosology of nervous system defects. II. BirthDefects 7: 33, 1971.

9. Sakuragawa, N., Sakuragawa, M., Kuwabara,T., et al.: Experimental model of Niemann-Pick disease: sphingomyelinase reduction in-duced by AY9944, Science, 1976 (in press).

10. Chen, I-Li, and Yates, R. D.: An ultra-structural study of opaque cytoplasmic in-clusions induced by Triparanol treatment,Am. J. Anat. 121: 705, 1967.


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