Menkes' kinky hair disease: a light and electron microscopic study of ...

Menkes' kinky hair disease: a light andelectron microscopic study of the eye

Shirley H. Wray,* Toichiro Kuwabara,* * and Paul Sanderson

Light and electron microscopic studies of the ocular tissue of a case of Menkes' kinky hairdisease are described. The copper deficiency responsible for this systemic and neurologicdisease appears to cause a progressive degeneration of retinal ganglion cells, loss of nervefibers, and optic atrophy. The pigment epithelium is also abnormal with only small andirregular melanin granules present among electron-dense inclusion bodies. Abnormal elasticais present in Bruch's membrane.

Key words: Menkes' kinky hair disease, ocular pathology, electron microscopic studyMenkes' disease, retinal ganglion cell degeneration.

I n 1962, Menkes and co-workers1 de-scribed a sex-linked recessive hereditarydisease characterized by early progressivepsychomotor deterioration, seizures, spastic-ity, hypothermia, pili torti, and character-istic facies. Later studies24 of this diseasedemonstrated bone changes resemblingscurvy and tortuosity of cerebral arteriesdue to fragmentation of the internal elastic

From the * Department of Neurology, Massachu-setts General Hospital; Howe Laboratory ofOphthalmology, Massachusetts Eye and EarInfirmary, Boston, Mass, and the "*"Laboratoryof Vision Research, National Eye Institute,National Institutes of Health, Bethesda, Md.

This project was supported in part by Grant No.NS 11551, awarded by the National Instituteof Neurological and Communicative Disordersand Stroke, Department of Health, Education,and Welfare, and in part by a grant from Fightfor Sight, Inc., New York City.

Submitted for publication June 27, 1975.

Reprint requests: Dr. S. H. Wray, Howe Labora-tory, 243 Charles St., Boston, Mass. 02114.

lamina. Similar vascular changes were alsofound widely in other arteries5 occasionallyaccompanied by occlusion. Menkes attrib-uted the disease to a metabolic defectwhose site he was unable to localize.

In 1966, O'Brien and Sampson0 per-formed lipid analyses on frozen brain tis-sue in two siblings with kinky hair diseaseand found significantly lower values in theproportions of docosahexaenoic acid, themost highly unsaturated fatty acid in thebrain, in the glycerophosphatides fromcerebral gray matter. This finding raisedthe possibility that the pathogenesis of thedisease might be due to the accumulationin neurons of lipid oxidation productswhich may inhibit the mitochondrial andmicrosomal enzyme system. Additional sup-port for this hypothesis had already comefrom Aguilar and co-workers2 who foundsudanophilic, acid-fast, and periodic acid-Schiff positive inclusions in the cytoplasmof the Purkinje cells.

A new approach to the etiology resulted

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Menkes' kinky hair disease 129

Fig. 1. Foveal zone of the retina. Number of the ganglion cells has been decreased pro-foundly. Other layers of the retina appear to be normal. Hematoxylin-eosin, xl50. Bars in-dicate one micron unless otherwise marked.

when Danks and co-workers'5' 7 noting thatcopper-deficient sheep have abnormal wooland similar arterial changes to the childrenwith Menkes' kinky hair disease, investi-gated copper metabolism in seven affectedbabies. They found abnormally low levelsof serum copper, copper oxidase, andceruloplasmin, and a defect in the intra-cellular transport of copper in the gut epi-thelium and the release of copper fromthese cells into the blood. He attributed thedefect in biogenesis of elastin and collagento a chronic copper deficiency state remi-niscent of the widespread connective tissuedisease in copper-deficient animals sincecopper, in the enzyme amino oxidase, isneeded to form the cross-linkages betweenlysine residues of elastin. A decreased ac-tivity of cytochrome oxidation has alsobeen demonstrated in kinky hair diseaseand an inadequacy of cytochrome oxidasemay result in a lack of high-energy phos-phate required for nerve cell maintenanceand function. Copper also has an importantrole in myelin formation.8 Thus, the extentto which each of these biochemical path-ways contributes to the neurologic deficitand to the pathologic changes is underextensive study.

Fig. 2. Flat preparation of the retinal blood ves-sels in the posterior zone. Capillaries are unevenin caliber. Several acellular strands are present(arrow). Periodic acid-Schiff and hematoxylin,x250.

Various therapeutic regimens have beentried to correct the copper deficiency. Re-cently, Lott and co-workerslJ have reportedsome success following the administration


130 Wratj, Kuwabara, and Sanderson Investigative OphthalmologyFebruary 1976

Fig. 3. Inner layers of the posterior retina. Inner limiting membrane (ILM) is thickened. Thearea is occupied mainly by Muller's cell cytoplasm. Large spaces (*) appear to be formedby degeneration of ganglion cells. Collapsed capillary wall is seen in the upper right corner(arrow) x 16,000.

of high-dose oral copper supplement in thepresence of L-histidene.

Studies in Australia have shown thatMenkes' disease is not rare and the clinical,neuropathologic, and biochemical featureshave already been reported extensively inthe literature. Nevertheless, the eye has re-ceived little attention. Neither Menkes andco-workers1 nor Bray10 noted any abnor-mality of the fundus in their cases eventhough the infants failed to follow a light,or showed constant horizontal nystagmus.Other observers- ai report a normal ap-pearance of the retina apart from possiblemild optic disc pallor or the suggestion oftortuosity of the vessels. In three additionalcasesa no eye examination is described.Billings and Degnan4 recorded an electro-retinogram (ERG) in their patient whothey suspected was blind. The fundi were

unremarkable but the infant, at age 13months, failed to fix or follow objects orrespond to menace. The ERG showed mod-erately decreased photopic beta-waves (ameasure of cone function) and almost noscotopic beta-waves (a measure of rodfunction) bilaterally. With a flash stimulusalmost no visually evoked response (VER)was recorded over the occiput. Similar re-sults have been documented in anothercase and an attempt made to correlate theabnormal ERG with serum copper levels.11

An absence of improvement in visual func-tion after elevation of the serum copper tonormal was noted. To our knowledge theeyes in these cases have not yet been exam-ined histologically.

Reports of the histopathologic changes inthe eye in Menkes' disease have in factbeen sparse and confined to light micro-


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Menkes kinky hair disease 131

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Fig. 4. Ganglion cells in the foveal zone. A, relatively normal cell. Mitochondria are markedlyswollen. B, degenerating ganglion cell. Nucleus is karyolytic. Rough endoplasmic reticulumis swollen ( °) . Both x 16,500.

scopic studies.1- The purpose of this paperis to report the ocular pathology visualizedwith the light and electron microscope inthe tissues of the eye of a three-year-oldboy in whom the diagnosis of Menkes1

kinky hair disease was established neuro-logically and biochemically prior to deathand in whom a detailed neuro -ophthalmicexamination was performed during thecourse of his illness.

Case Report

G. B. (NEI; LVR E208), a white boy 3.3-years-old was the 5 pound, 14 ounce product oran eight-month pregnancy. He was the youngestof seven children. He had five normal siblings,aged 4 to 14 years, and one male sibling whodied 10 years earlier at the age of 16 monthswith Menkes' disease.

The infant's early development was slow andfrequent seizures began at age two months.Initially these consisted of intermittent twitchingof the left-sided limbs, rolling back of the eyes,cyanosis, and salivation. Each seizure lasted only

a few minutes. An electroencephalogram (EEG)showed random high voltage 2 to 3 cycles persecond activity over the entire left hemisphere andrandom sharp waves over the left temporal lobe.He was treated with phenobarbital. The patienthad extremely light, sparsely distributed, coarsestubbly hair and eyebrows. The face was palewith puffy cheeks and micrognathia. No bonyabnormalities were noted.

By age five months mental retardation wasevident. A biopsy of the right cerebellum andright posterior temporal tip was performed andrevealed marked loss of neurons and intenseastrogliosis. The serum copper level was 20 me.per cent (normal 75 to 160), ceruloplasmin 2.0mg. per cent {normal 15 to 35), urinary copper18.5 meg. per 24 hours. Blood amino acids werenormal.

The neuropathologic study together with theevaluation of the serum and urine copper levelsestablished the diagnosis of Menkes' kinky hairdisease and the patient received parenteral ad-ministration of trace elements including zinc, cop-per, magnesium, and iodine. After several weeksof therapy the serum copper level was 29 meg.per cent, ceruloplasmin 20 mg. per cent. Thehematocrit ranged between 35 and 41 per cent.


132 Wray, Kuwabara, and Sanderson Investigative OphthalmologyFebruary 1976

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Fig. 5. Photoreceptor cells. Mitochondria of theinner segment (IS) are markedly swollen. Lamel-lar membranes of the outer segments are relativelywell preserved. xl6,500.

The neurologic signs of a progressive degenerativedisorder continued unchecked. Angiographic studyof several arteries showed minute changes whichwere considered to be secondary in nature.

At age 2.10 years, five months before death,a neuro-ophthalmic examination was performed.The eyes showed the characteristic roving move-ments of a blind child. The patient did not blinkor cry in response to bright light or to menaceand failed to fix on or follow a light or toy. Nonystagmus could be elicited with the opticokineticdrum. The irides were blue and did not transil-luminate. The cornea and lens were transparent.The pupils were equal and only just reactive tolight, equal bilaterally. Roving eye movementsshowed a full range of ocular motility and therewas normal horizontal ocular deviation on rota-tion. There was no nystagmus. The fundus showeda blond appearance similar to that of albinismwith prominent visibility of the choroidal vessels.The macula had a normal foveal reflex but slightmottling of the pigment epithelium was presentjust temporal to the fovea in both eyes. Theretinal blood vessels were normal. The opticnerves showed marked optic atrophy. Both the

discs were normal in size and configuration, uni-formly pale, and almost chalk white in color. Incontrast the retina of the posterior pole lookeda "healthy pink" suggesting widespread loss ofnerve fibers from the nerve fiber layer.

Pathologic study. The eyes were enucleatedabout five hours after the death of the patient.One eye was fixed in 10 per cent neutral formalinfor general histologic study. The central hori-zontal sections were stained with hematoxylin-eosin, periodic acid-Schiff reaction and Bodian'scombined silver, and Luxol fast-blue staining.A small portion of the posterior retina was em-bedded in gelatin and the frozen sections stainedwith Oil red O. The other eye was opened atthe pars plana with a small slit cut and fixedin 4 per cent glutaraldehyde solution at roomtemperature for a few days. Small pieces of thecornea, trabecular meshwork, ciliary body, opticnerve, and several locations of the retina wereexcised. They were postfixed in 1 per cent osmiumtetroxide for one hour, dehydrated in ethylalcohol, and embedded in an epoxy resin. Sectionswere cut 0.5 fi thick, stained with toluidine blue,and examined light microscopically. The ultrathinsections were stained doubly with uranyl acetateand lead citrate and examined by electronmicroscopy.

Histologic findings. The size of the globe andproportions of each component of the eye arenormal. The cornea, sclera, and ciliary bodyappear to be normal. The iris epithelium ismarkedly vacuolated, apparently due to inter-cellular separation. The lens tissue at the bowand the anterior zone is moderately vacuolated.The lens epithelium and the capsule show noabnormality. The angle and the trabecular mesh-work also appear to be normal except for somepigmentation in the trabecular cells. Althoughthe retina shows normally formed layers andfoveal structure, the number of ganglion cellsare markedly decreased. This is strikingly evidentin the macula zone where only a single cell layerof ganglion cells is preserved (Fig. 1). Numerousglia cells are present in the ganglion cell andnerve fiber layers. The surviving 'ganglion cellsare vacuolated but no pathologic substance hasaccumulated. The pigment epithelial cells areirregular in size and appear less pigmented thannormal. The choroidal tissue is free from in-filtrating cells. Both retinal and choroidal bloodvessels appear to be normal histologically. How-ever, flat preparations of the retinal vessels, inthe general area, show numerous acellular strands(Fig. 2).

The optic nerve is atrophic. The disc surfaceis covered with proliferated glia cells and theseptal connective tissue is considerably thicker.A myelin stain reveals marked demyelination ofthe nerve particularly in the peripheral bundles,


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Fig. 6. Optic nerve. Number of the myelinated fibers is markedly reduced. Myelin sheathsof individual fibers are thinner than normal. Some are degenerating. Microtubules are ab-sent in the axon. A nucleus of the astrocyte is seen in the lower left corner. The astrocyticcytoplasm is markedly increased. x8,000.

and a Silver stain shows considerable loss ofnerve axons. The remaining nerve fibers showa bead-like structure. Glia cells are increased pro-foundly in both the intraocular and retrobulbarzones. No fat droplets have accumulated in thecells of the optic nerve. The changes in the opticnerve are subsequent to the death of the retinalganglion cells.

Electron microscopy. Since the material wasobtained a few hours after the death of the pa-tient and stored in the fixative for a prolongedtime, preservation of the cytologic detail is notideal. The retinal tissue shows a considerable de-gree of postmortem change with swollen mito-chondria and vacuolation in many cells. How-ever, several meaningful findings are observed inthe eye,

As the ganglion cells have decreased, the innerlayers of the retina are replaced by increasedcytoplasm of Miiller's cell (Fig. 3). The remain-ing nerve fibers contain fine neurofibrils insteadof microtubules. The surviving ganglion cells inthe macula zone still maintain characteristic micro-organelles. However, their mitochondria, roundendoplasmic reticulum, and Golgi apparatus aremarkedly swollen, and some cells also show de-generating changes (Fig. 4, A). The cytoplasmof these cells is edematous and the nucleus is

karyolytic (Fig. 4, B). Although cell componentsin the inner plexiform layer are relatively wellpreserved, no synaptic organs are demonstratedin the sections. Also the outer synaptic organsare poorly preserved. Cone synapses have changedinto large bodies containing amorphous sub-stance. The pedicles have no interdigitating hori-zontal cell components, synaptic vesicles or rib-bons. Nuclei of the photoreceptor cells are rela-tively well preserved and their number appearsto be within the normal range. Many mito-chondria of the inner segments are markedlyswollen (Fig. 5). The rod outer segments aremostly disarrayed but their individual disc mem-branes appear to be normal. The cone outer seg-ments, especially in the macula zone, show ir-regularly arranged membranes. The retinal capil-laries seem to be normal, but some vessels havea slightly tortuous basement membrane withuneven thickness. Their endothelial cells showan increased number of rough endoplasmic retic-ula. The cytoplasm of the mural cells of thesecapillaries is considerably electron dense.

The optic nerve is atrophic. The number ofmyelinated nerve fibers is markedly reduced. Thetissue is occupied by astrocytes which have in-creased their number. The remaining nerve fibershave relatively thin myelin sheaths and the micro-



Fig. 7. Pigment epithelium in the posterior zone. The cytoplasm contains numerous electrondense inclusion bodies. Only a few melanin granules are seen in this area. Appearance ofthe micro-organelles is normal. x 10,000.

Fig. 8. Bruch's membrane in the posterior zone. Elastica is small and scattered irregularly(arrows). Fine fibril substance is forming banded masses. xl8,000.

tubules are missing (Fig. 6). No abnormal ma-terial has accumulated in the optic nerve.

The pigment epithelial cells show reductionof melanin granules. Large spindle-shaped melaninbodies, which are commonly present in the apicalportion of the cell and in the microvilli, are ab-sent. Only small and irregular melanin granulesare present among electron-dense inclusion bodies

(Fig. 7). Lipofuscin particles are also abundant.Mitochondria contain small electron-dense par-ticles within the matrix. Other micro-organellesappear to be normal. Apicolateral junctions arenormal and the epithelial cells are firmly at-tached to each other. The finding of sparse finemicrovilli may be due to postmortem change.Vesicular substance, which is present in the sub-


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P

0.5H

• \ •

Fig. 9. Higher magnification of Bruch's membrane in the posterior zone. Banded masses show1,000 A spacing. Elastica is small and sparse. x48,000.

retinal space, appears to be broken pieces ofthe fine microvilli of the pigment epithelium.The basal infoldings are somewhat widened, butthe basement membrane is uninterrupted.

The structure of Bruch's membrane is ab-normal. The most striking change is the scantyand irregular distribution of the elastic membrane(Fig, 8). Only small pieces of well-formedelastica are present within Bruch's membrane.The distribution and number of collagen fibersappear to be normal. Fine fibrils, which arenormally seen around the elastic membrane, areirregularly distributed, They often aggregate intobanded masses (Fig. 9). These bandings areidentical to those of 1,000 A collagen fibers,which are seen regularly in Bruch's membrane insenescence. The basement membrane of thechoroidal endothelium appears to be normal.However, there are several locations where thebasal portion of the endothelial cell extends intoBruch's membrane.

The corneal epithelium, Bowman's membrane,Deseemet's membrane, and the major part of the

stroma appear to be normal. However, the super-ficial stroma, including the central zone, showseveral small foci in which lamellar arrangementof the collagen fibers is irregular and fine fibrilshave been increased (Fig. 10). These findingsare similar to those of the normal limbus or ofa scar. No deposit of abnormal substance ispresent in Deseemet's membrane. The trabecularmeshwork is normally developed. The trabeculaecontain numerous well-developed elastic tissuewhich is surrounded by fibrils. Normal collagenfibers are scanty in the trabecula (Fig. 11).Melanin particles and other inclusion bodies arepresent abundantly in the trabecular endothelium.The Schlemm's canal is also well developed. Theendothelial cells show their normal giant vacuoles(Fig. 12). No appreciable changes are noticedin the ciliary body.

Discussion

The present case shows all the clinicaland pathologic characteristics of Menkes*


136 Wraij, Kutoabara, and Sanderson Investigative OphthalmologyFebruary 1976

Fig. 10. Central cornea. Lamellar arrangement ofthe collagen fibers are missing. Fine fibrils aremarkedly increased. xl5,000.

kinky hair disease. As Danks and asso-ciates5' 7 have shown, this disease is be-lieved to be caused by a generalized cop-per deficiency in the body and the lowcopper levels in cells and tissue fluid ap-pears to seriously interfere with certainenzyme systems and the maintenance ofneural cells and hair.

Light and electron microscopic studiesof the ocular tissue of this case have dem-onstrated interesting findings. The develop-mental process of the eye appears to be nor-mal, since the size of the globe, thicknessof the retina, and the diameter of the duramater of the optic erve are normal for theage of the patient. The most striking patho-logic change is a degeneration of the gan-glion cells with loss of nerve fibers andatrophy of the optic nerve. The ganglioncell appears to degenerate first and signs ofdegeneration are seen in some remaining

Fig. 11. Trabecular meshwork, Elastica in thetrabeculae appears to be normal (arrows). Theendothelial cell contains melanin pigments.x21,000.

ganglion cells of the macula zone at thetime of death. The pathologic processseems to be slowly progressive and thenecrotic cell debris has been carried outfrom the retina tissue so that no accumu-lation of specific substance is noted withinthe retina and optic nerve. Also no phago-cytic or inflammatory reaction is presentin the eye. These findings are somewhatdifferent from those of the central nervoussystem.

The mitochondria of the surviving gan-glion cells, photoreceptor inner segments,and of other neural cells of the retina are


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Menkes kinky hair disease 137

markedly swollen and an electron-densesubstance is present in the matrix of themitochondria in the pigment epithelium.These mitochondrial changes may be sig-nificant in light of the demonstration of thedeficiency in cytochrome oxidation in thisdisease but, since swelling of mitochondriais the most common postmortem change, itis difficult to definitely correlate thesefindings to a dysfunction of the cytochromeoxidation system.

The photoreceptors are not involved di-rectly in this disease. Lamellar membranesof the outer segments as well as the mainphotoreceptor cells are well preserved.Marked swelling of the synaptic pediclesmay be due to postmortem change.

The metabolic deficiency of copper ap-pears to have a direct effect on mainte-nance and structure of hair since tyrosinase,a copper containing enzyme, is needed forthe synthesis of melanin. Similarly, coppermay play a role in melanin formation inother pigmented cells. The distributionand amount of melanin granules of thepigment epithelium are uneven. The cyto-plasm contains smaller and irregularlyshaped melanin particles. Numerous elec-tron-dense inclusion bodies and lipofuscingranules are abundant in the cytoplasm.These changes seem to correlate with thepathologically pale funduscopic appearancein this child. The abnormal pigmentationin the trabecular meshwork may be due tophagocytosis of degenerated melanin pig-ments within the eye.

The changes in Bruch's membrane areinteresting and noteworthy. The elasticmembrane of Bruch's membrane is welldeveloped at the time of birth. Althoughit is not clear whether the pigment epithe-lial cell or endothelial cell of the choroidalcapillary is involved, the elastica formationof Bruch's membrane of this case appearsto be considerably suppressed. The amountof the elastica is significantly small and itforms small patches. The fine fibrils whichare normally present around the elasticaaggregate into large banded structures.Pathologic changes of the elastic tissue of

Sch

0,

Fig. 12. Schlemm's canal. The endothelial cellshows a normal giant vacuole (*) . Normal col-lagen fibers are sparse in the gound tissue ofthe meshwork. This area contains mainly finefibrils. x45,000.

the blood vessels have been reported inother organs and it seems that a similarpathogenetic process in elastica formationmay be occurring in Bruch's membrane.The retinal capillaries show slight patho-logic changes and the flat preparationsdemonstrated several acellular strands andan uneven caliber of the vessels. Thesechanges may however be nonspecific.

Changes in the cornea may also be non-specific with scarring secondary to ex-posure. Although the lamellar arrangementis not quite normal in general, the cornealcollagen fibers appear to be well devel-oped.



We thank Dr. Janowska and Dr. Kolodny forpermission to publish this case, D. Player fortechnical assistance, and Miss M. Saunders fortyping the manuscript.

REFERENCES

1. Menkes, J. H., Alter, M., Steigleder, G. K.,et al.: A sex-linked recessive disorder withretardation of growth, peculiar hair andfocal cerebral and cerebellar degeneration,Pediatrics 29: 764, 1962.

2. Aguilar, M. J., Chadwick, D. L., Okuyama,K., et al.: Kinky hair disease. I. Clinical andpathological features, ]. Neuropathol. Exp.Neurol. 25: 507, 1966. '

3. Wesenberg, R. L., Gwinn, J. L., and Barnes,G. R., Jr.: Radiological findings in the kinky-hair syndrome, Radiology 92: 500, 1969.

4. Billings, D. M., and Degnan, M.: Kinky hairsyndrome: a new case and a review, Am. J.Dis. Child. 121: 447, 1971.

5. Danks, D. M., Campbell, P. E., Stevens,B. J., et al.: Menkes' kinky-hair syndrome.An inherited defect in copper absorptionwith widespread effects, Pediatrics 50: 188,1972.

6. O'Brien, J. S., and Sampson, E. L.: Kinkyhair disease. II. Biochemical studies, J. Neu-ropathol. Exp. Neurol. 25: 523, 1966.

7. Danks, D. M., Campbell, P. E., Walker-Smith, J. et al.: Menkes' kinky-hair syn-drome, Lancet 1: 1100, 1972.

8. Everson, G. J., Schrader, R. E., and Wang,T. I.: Chemical and morphological changesin brains of copper-deficient guinea pigs,J. Nutr. 96: 115, 1968.

9. Lott, I. T., DiPaolo, R., Schwartz, D., etal.: The pathogenesis and treatment of cop-per deficiency in steeley hair syndrome.Abstract of the ANA meeting, Arch. Neurol.32: 356, 1975.

10. Bray, P. F.: Sex-linked neurodegenerativedisease associated with monilethrix, Pediatrics36: 417, 1965.

11. Levy, N. S., Dawson, W. W. Rhodes, B. J.,et al.: Ocular abnormalities in Menkes'kinky-hair syndrome, Am. J. Ophthalmol.77: 319, 1974.

12. Seelenfreund, M. H., Gartner, S., and Vinger,P. F.: The ocular pathology of Menkes'disease, Arch. Ophthalmol. 80: 718, 1968.


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