Journal of Neuropathology and Expezimental Neurology Vol. 55. No.3 Cop yright 't"J J996 by the American Assoc iation of Neuropathologists March. 1996

pp. 348-356

Ependymal Changes in Sudden Infant Death Syndrome

JOAQuiN LUCENA, MD AND FEux F. CRUZ-SANCHEZ, MD, PHD, MRCPATH

Abstract. The ependyma may befall a variety of pathogenic noxae during fetal life. resulung in histological changes which may pers ist after birth but arc without clinical manifestations. Eight of a senes of 19 children who died suddenly and unexpectedly, where no explanation as to the cause of death wa s found at autopsy. were shown to have diverse histological features involving the ependyma. Five IJ.m paraffin-embedded brain tissue sections including frontal. tem­poral. oc cipital, and ventricular horns as well as the fourth ventricle were stained with hematoxylin-eosin (H&E) and luxol fast blue (LFB) . Immunohistochemical stams using antibodies to glial fibrillary acidic protein (G FAPl. virnentin and Sol 00 were also performed, Findings included areas of denuded and/or desquamated ependyma. rosettes in different stages of formation. vacuoles and/or pseudocysts, inflammatory changes consisting in macro- and microglial nodules in the subependyrnal layer. and gliosis. Chronic brain edema was seen in 4 cases. Our findings indicate that ependymal changes in sudden infant death syndrome (S IDS) cases belong to the prenatal or early postnatal period, thus providing. indirectly. a morphological substrate for the previous existence of a noxa that may also affect other CNS areas. and thus being in the position to produce cardiorespiratory control dysfunction .

Key Words: Ependyma: Neuropathology; Sudden infant death syndrome.

INTRODUCTION

The ependyma is an epithelial lining of neuroecto­dermal origin which borders the ventricular system in­cluding the central canal of the spinal cord . Severa) noxae may affect the ependyma and adjacent tissue both during gestation and al so subsequently. Changes of ventricuLar volume and/or ischemic and inflamma­tory processes and toxic agents may lead to structural changes, and it is known that the ependyma of humans and other mammalians does not regenerate at any age (I). Many pathological processes exert their effects during fetal development , producing some structural changes which may remain after birth even though clinically silent. At times, some of the resulting histo­logical changes may be pointers to the original disease process.

Sudden infant death syndrome (SIDS) is defined as "the sudden death of an infant under one year of age which remains unexplained after a thorough case in­vestigation, including performance of a complete au­topsy, examination of the death scene and review of the clinical history" (2).

Current hypotheses suggest that SIOS results from abnormalities of central nervous system (CNS) struc­tures regulating respiration and cardiovascular activity during sleep (3, 4). These abnormalities have been re-

From the Neurological Tissue Bank, Hospital Clfnico-Universiry of Barcelona (FCS) and Anatomical Forensic Institute. Barcelona (l L) .

This study was supported by the BIOMED-! program (PL931359) from the Commission of the European Communities. by a framework a~ment between Carburos Met:ilicos S.A. and Hospital Clinic for research in brain banking and by Comissto Interdepartarnental Per Re­cerca i Tecnologia (CfRIT) Generalitat de Catalunya.

Correspondence to: Dr FF Cruz-Sanchez. Banco de Tejidos Neurol­6gicos. Servicio de Neurologia, Hospital Clfnico, Villnrroel 170. 08036 Barcelona. Spain.

lated 10 impairment in brain development mainly as­sociated with maternal and environmental conditions traditionally known to be risk factors (5). However, in most instances, morphological findings confirming a particular association are found lacking .

Subtle tissue changes in various organs including the CNS have been described in SIDS (5). Increased brain weight (6), white matter lesions (7, 8) , and brainstern gliosis (9. 10) have all been reponed, but ependymal changes have not. None of the histological findings have ever been thought sufficient to explain SIDS deaths; however, tissue findings can be an indication of the impairment of several pathophysiological mecha­nisms involved in normal eNS development (5).

The present study describes pathological changes af­fecting the ependyma in 8 sros cases collected ac­cording LO a recently published research protocol (11). Similar findings have, to our knowledge, not been re­ported. These results further add to the hypothesis that some neuropathological changes found in SrDS are shared with those of other described developmental brain disorders (12) and dysfunctions (5).

MATERIALS AND METHODS

Twenty-eight infants who died unexpectedly between 1991 and 1994 were studied from a pathological point of view. including a detailed neuropathological examination. Age ranged from 19 to 330 postnatal days; 20 were males and 8 females. Death was explained in 9 cases (32%) (I I). The remaining 19 cases (68%) showed clinical. pathological , and medico-legal diagnost ic criteria of SIDS (2).

Brains were removed shortly after death and fixed in a 10% formaldehyde solution for 3 weeks (w) . The average postmortem delay was 14 hours (h). Subsequently, brains were cut coronally, sampled. and examined histologically ac­cording to a standardized protocol (l I). Eight cases (42%) showed diverse pathological features involving ependyma

348

349 THE EPENDYMA IN srns

TABLE I Some Clinical and Morphometrical Data of 8 SillS Cases

Bilth Moth. Head Head Brain Age Deliver weight APGAR Gesta­ age eire. eire. weight

N days. weeks g score tion years em (I) cm (2) g

I f 83 35 twin 1,160 g 6-8 2nd 26 yr 29 41 479 g 2 m 223 39 .2,800 g 8-10 3rd 36 yr 47 970 g 3 m 64 40 39 650 g 4 f 62 38 2,900 g 8-10 l st 27 yr 38 670 g 5 f 163 36 2,600 g 7-9 3rd 34 42 680 g 6 m 54 40 3,120 g 10-10 8th 38 yr 37 39 507 g 7 m 49 38 3,250 g 9-10 4th 32 yr 34 39 574 g 8 m 165 37 3,030 g 10-10 1st 22 yr 32 41 746 g

N: number of case and sex; f: female; m: male. Age: age at death. Deliver: gestation age at delivery. Gestation: number of pregnancies. Moth. age: age of the mother. Head eire (I): head circumference at birth; Head eire (2): head circumference at death.

TABLE 2 Ependymal Histological Features in 8 SillS Cases

Histological features 2 3 4 5 6 7 8

Ependymal epithelium Denuded ependyma + + ++ ++ Desquamated ependyma ++ + ++ + ++ ++

Hypertrophy

Rosettes +++ ++ ++ +++ ++ + Demiroseues ++ ++ ++ Ependymal rests +++

Subependymal layer Reactive or inflammatory changes

Microglial nodules ++ ++ ++ ++ Macroglial nodules ++ ++ ++ Gliosis ++ ++ ++ ++ ++ +++ ++ Residual germinal matrix + ++ +++ Perivascular infiltrates + Hemosiderin-laden macrophages + + ++

Non-inflammatory changes Vacuolation + ++ ++ ++ ++ Pseudocysts ++ + ++ + ++

Other CNS changes

Chronic edema ++ ++ +++ ++ Old subarachnoidal hemorrhagic

changes ++ Microglial nodules +

-: absent; +: mild or scant)'; + +: moderate; + + -t-: severe or abundant.

and were further studied. These cases were compared to 6 the ABC complex (all of them from Dako) were also per­controls whose death was explained but where the CNS was formed. not involved. Clinical data of these 8 cases are shown in Table I. RESULTS

Five urn sections of paraffin-embedded brain tissue from In spite of the absence of macroscopical abnormali­

frontal, temporal, and occipital regions including ventricular­ties, including hydrocephalus, SIDS cases showed sev­horns and the fourth ventricle region were stained with he­eral histological changes involving the ependyma whichmatox ylin-eosin (H&E) and luxol fast blue (LFB). Immu­are summarized in Table 2. Most changes were found innohistochemical stains using polyclonal antisera for the glial

fibrillary acidic protein (GFAP, I :200) and S- 100 protein (S­ frontal and occipital horns. Controls did not show sig­100. I :500), a monoclonal antibody for virneruin (I: 100). and nificant changes affecting ependyma. Table 3 shows the

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350 LUCENA AND cRuz-sANcHEZ

TABLE 3 Topographical Distribution of Histological Features in Ependyma of 8 SillS Cases and 6 Controls (C'I'L)

Frontal hom Temporal hom Occipital hom Fourth ventricle

Features SillS cn SillS en SillS cn SillS cn Denuded ependyma 4/8 ·0/6 0/8 0/6 4/8 0/6 [/8 0/6 Desquamated ependyma 5/8 0/6 1/8 0/6 4/8 0/6 0/8 0/6

Rosettes 4/8 0/6 2/8 0/6 4/8 t/6 3/8 0/6 Demirosettes 2/8 0/6 2/8 0/6 2/8 0/6 0/8 0/6 Ependymal rests 0/8 0/6 118 0/6 0/8 0/6 0/8 0/6

Microglial nodules Macroglial nodules

4/8 2/8

0/6 0/6

1/8 1/8

0/6 0/6 .

3/8 3/8

0/6 0/6

0/8 0/8

0/6 0/6

Gliosis 7/8 2/6 5/8 1/6 5/8 0/6 1/8 0/6 Residual germinal matrix 3/8 2/6 0/8 1/6 2/8 t/6 0/8 0/6 Peri vascu lar infiltrates 1/8 0/6 0/8 0/6 0/8 0/6 0/8 0/6 Hemosiderin-laden rnacrophages 3/8 0/6 0/8 0/6 t/8 0/6 0/8 0/6 Vacuolation 5/8 2/6 2/8 116 4/8 t/6 0/8 0/6 Pseudocysts 5/8 0/6 1/8 · 0/6 3/8 0/6 0/8 0/6

Immunohistological findings

GFAP 1/8 2/6 1/8 1/6 0/8 1/6 1/8 0/6 Vimentin 7/8 3/6 5/8 3/6 5/8 4/6 4/8 2/6 5-100 8/8 6/6 8/8 6/6 8/8 5/6 6/8 5/6

regional distribution of histological features including immunohistological findings in SIDS and controls.

In 4 cases, some areas of the ventricular surface ap­peared denuded, probably due to necrosis of epithelial cells (Fig. IA). In addition, 6 cases showed ependymal cells separate from the ventricular wall (Fig. IB). In most cases, these changes were accompanied by ro­settes, macro/microglial nodules and gliosis in the su­bependymal layer. The presence of ependymal rosettes was a common feature. It was observed in different stages of their formation (Fig. 2). In some cases, ro­settes formed sequestration and/or diverticuli of the ependymal surface. In other cases rosettes were derived from a space of discontinuous ependyma which formed a loop penetrating into the cerebral parenchyma. In 3 cases, demi-rosettes often appeared as a row of sub ­ventricular ependymal cells parallel to the surface (F ig. 3). Red blood cells, fibrin , and lymphocytes (l case) filled the central space.

In one case, 'abu ndan t immunohistochemistry­negative "curled" ependymal rests were observed in the white matter surrounding the lateral ventricle of the temporal horn, which were labeled " displaced epen­dymo-choroidal epithelial clusters" (Fig. 4) .

Ependyma from SIDS and controls showed irnmu­noreacti vity for S- tOO and vimentin (Fig . 5) ~ $-100 ex­pressed a cytoplasmic and nuclear pattern of immune­staining while vimentin showed a somatic pattern of positivity with perinuclear accentuation. Few epithelial cells showed somatic pattern of GFAP immunostaining (Fig. 6).

Micro- and macroglial nodules and gliosis of the sub-

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ependymal layer (Fig. 7) were observed. Some GFAP­positive glial nodules were of small size and located under the ependyma, occasionally protruding from and through the denuded spaces (Fig. 8) . Other nodules ap­peared larger and were composed of macroglial cells and were surrounded by GFAP-positive reactive astro­cytes. In most cases, glial proliferation caused thicken­ing of the germinal matrix and appeared to be GFAP positive (Fig . 6A). In one case, very few peri ventricular small vessels surrounded by mononuclear cells were

·seen. Hemosiderin-laden macrophages were fo und in three cases in the gliolic zone. In ' j cases, there was tis sue rarefaction of the 'subependymal white matter forming vacuoLes or spongiform changes. In 5 cases, large spaces resembling pseudocysts were also found. A degree of vacuolation up to the production of desqua­mation and stripping of the ependymal surface were seen (Fig. 9). Another pathological feature affecting the remaining central nervous system tissue was chronic edema (3 cases) with the presence of large astrocytes (Fig. 10), some with intracytoplasmic vacuoles and "lip­idization." One case showed arachnoidal fibrous thick­ening and hemosiderin-laden macrophages. In one case, very few microglial nodules were seen in the white mat­ter of the frontal part of the centrum semiovaie .

DISCUSSION

Previous studies have described two main categories of findings at the level of the eNS in SIDS, e.g, " de ­layed maturation" and .. solely pathological. " Matura­tion defects ranged from high dendritic spine density in neurons from hypoglossal nuclei (12), delayed ce­

351 THE EPENDYMA IN SIDS

Fig. I. Ependymal surface in SillS cases: A (Top) . denuded ependyma (Case I, frontal hom). B (Bortorn). Ependymal cells separated from the brain tissue surface (desquamation) (Case 5, occipital hom). A: HE X200, B: HE X400.

rebral myelination (8), to atrophy of arcuate nuclei (13) and thickening of the cerebellar external granular layer (II, 14). Pathological changes such as gliosis (10), mi­croglial proliferation (IS), and peri ventricular and sub­corticalleukomalacia (7) have been related to different causes (5, 11) . Furthermore, neurochemical studies in SIDS have demonstrated neurotransmitter changes in the brainstem (16) which may have its morphological counterpart (5, 12, 17).

Ependymal changes observed in our cases were of some standing and consisted of a whole encompassing spectrum from damage to the ependymal epithelium to rosette formation. Denuded and desquamated ependy­ma, rosette and derni-rosette formation, ' thickening of subependymal layer with glial proliferation. as well as features consistent with previous hemorrhage are' pathological changes which may disturb the effective circulation and reabsorption of cerebrospinal fluid (CSF) (18) and/or alter the secretory function of epen­dyma in fetal life (1). Some changes could be related

Fig. 2. Different stages in ependymal rosette formation (Case 3, frontal born): A (Top). Loop formation which pene­trates into the cerebral parenchyma (HE x6(0). B (Center). Sequestration of the surface ependyma (HE X400). C (Bottom). Discontinuous ependyma and rosettes in the subependyrnal lay­er: (HE X 400) .

to the increase in CSF voLume and/or pressure, leading to desquamation and to denuded ependyma, which may affect the normal milieu between ventricles and cere­bral parenchyma (19, 20). However, ventriculornegaly

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352 LUCENA AND cRuz-sANCHEZ

Fig. 3. Ependymal derniroserte showing lymphocytic infil­trates in the open side space (upper) (Case 6, temporal hom). (HE x 400).

was not observed in any of the cases studied. Edema and vacuolation of the subependyrnal layer could be representative of degrees, or be the forerunner to the desquamation of the ependyma. Further studies of CSF in SIDS and controls, such as protein estimation and others, may be of value in the recognition of abnor­malities in the flow of ventricular fluid and its com­position .

Other mechanisms lending support to our f ndings include ventricular hemorrhage in early postnatal life

.as well as viral infections of the mother during preg­nancy, causing damage to ependymal cells and sube­pendyrnal tissue with the formation of micro/macrogli­al nodules and gliosis (18) . Most changes observed in our cases may be markers of pathological processes occurring in the perinatal period. Subventricu lar glial nodules may be the end result of the damage from a variety of causes (I). Residua of germinal matrix. hemosiderin-laden rnacrophages, and periventricu lar pseudocysts are pathological features related to focal, subependymal hemorrhage probably occurring in the perinatal period (21) . Ependymal rosettes and derni­rosettes are secondary to ependymal damage (1. 20) and ependymal rests are rare features explained by the aberrant differentiation of some cells of the matrix lay­er (22).

Immunohistochemistry has been thought to be a suit­able tool for the study of phenotypic differentiation of eNS tissue (23) and some types of brain tumors (24), thereby linking the an tigenic expression to ontogenesis (25). However, in the case of immature tissue and/or embryonal tumors such a link is difficult to substantiate (26, 27) . Despite the fact that some authors have found a possible link between the ependymal expression of virnernin to pathophysiological mechanisms of some

J Neuropaihol £rp Neural. Vol 55. March. 1996

Fig. 4. Displaced ependyma-choroidal epithelial rest or cluster in the cerebral white matter. (Case 7, temporal lobe) (GFAP x 100).

congenital disorders, (28) our results are inconclusive; the only positive assertion that can be made is that S 100 is a specific marker for the ependymal line, in agreement with other authors who studied ependymal­derived tumors using the same antibody (29) . Epen­dymal remnants were consistently negative with all an­tibodies used, thus suggesting that they may represent the forerunners of ependyma, e.g. a preceding step in the ontogenesis .

Ependymal epithelium-like choroid plexus epithelium has absorptive properties (30). Its differentiation de­pends on an inductive action of the primitive ventricular neuroepithelial cells from the neural rube. The findings of GFAP expression in some epithelial cells could be explained on the basis of these concepts in a similar way of GFAP expression in choroid plexus cells (31). GFAP expression in ependyma and choroid plexus cells could be related to the ontogeny of the epithelium and/or to the product of degeneration in relation to age or to the type of cerebral pathology (edema, infarct, hemorrhage) . Assembling of GFAP in the same intermediate filaments has also been found using immuno-electronmicroscopy (32).

Features related to chronic edema were present in some cases. The presence of large hypertrophic astrocytes (some of them showing cytoplasmic vacuoles and lipid accumulation) is evidence of a reaction to brain damage (15, 33), including edema secondary to hypoxic mecha­nisms (34).

Astrocytes could phagocytose myelin or may be laden with lipids of yet unknown origin (35, 36) . Some authors have suggested that lipid-containing cells in infant brains may be the result of white matter damage in the periven­tricular region (37).

Very few histopathological features may be secondary

353 THE EPENDYl\1A IN SIDS

A

B

Fig. 5. Irnmunosrainings for 5-100 (A) and virnentin (B). Note the intense immunoreactivity of the ependymal epithelium for both antibodies showing a nuclear and somatic pattern for S-loo (arrows) (A) and a somatic pattern with perinuclear accen­tuation for virnentin (arrows) (B). (Case 8, temporal hom). A and B: X4oo .

to an acute process (see case 7) which is probably related to an infection at an ear ly stage of development. How­ever, this case shared other changes such as chronic ede­ma. vacuolation, and pseudocysts, which could explain­chronic evolution of the initial morbid processes.

Ependymal changes in SillS were compared with 6 controls which did not show significant features affecting ependyma. Using a similarly sized group of controls is

difficult. SIDS is the first cause of postneonataJ infant mortality in developed countries. Deaths by accidents in the first year of life are. fortunately, very rare in devel­oped countries and they are frequently related to intra­cranial damage. On the oilier hand, pediatric clinical au­topsies are scarce in the first year of life.

Neurological tissue banks facilitate such research as in the research of other neurological disorders (38). In

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354 LUCENA AND CRUZ-SANCHEZ

Fig. 6. Immunostainings for GFAP showing intense positivity in few epithelial cells (arrows) and in the germinal matrix (bottom in the figure) (A) as well as in the subependymaJ layer (B). Note the presence of some hypertrophic astrocyies (large arrows). (Case 8, frontal hom). A and B: X400.

Europe. a brain bank network has been developed (0

serve as a bridge between clinicians and neuroscientists in the study of etiology and pathogenesis of central ner­vous system disorders (39). One of the objectives of this action is to acquire and adequately preserve brain tissue for SIOS research (II). Nevertheless, finding adequate controls is one of the most crucial difficulties in neuro­pathological SIDS research.

j Neuropatbol Exp Neural. Vol 55. March, 1996

SIDS has been considered a sleep-related disorder of cardiorespiratory control (3). Currently. it is thought that prolonged episodes of apnea during sleep are the final pathway resulting in these dramatic deaths (40). The age at death in SillS (90% under 6 months) suggests an im­portant underlying mechanism involving growth and de­velopment (41).

In the first 6 postnatal months, the nervous system

355 TIIE EPENDYMA IN SIDS

Fig. 7. Macro-microglial nodule into the subependyrnal layer. (Case 2, frontal hom) (HE X200).

Fig. 8. Two GFAP·po.siove glial nodules protruding into a denuded ependymal space. (Case 3, occipital hom) (GFAP >(600) .

shows important maturative changes which are related to cardiorespiratory control (5); thus, SIDS occurs in a vul­nerable postnatal period for the CNS. Epidemiological risk factors in SIDS indicate a non-optimal intrauterine environment supporting the hypothesis that brain dys­function is originated in utero (5, 41). Other authors sug­gested prenatal virus infection (11, 16, 42). Variend and Pearse (43) observed that microglial nodules in a group of SIDS cases were related to an unrecognized intrauter­ine cytomegalovirus infection. Experimental studies dem­onstrated the potential for viruses to induce defects on the developing nervous system even with minor or clin­ically inapparent maternal or neonatal infection (20). Our findings further support these hypotheses , indicating the presence of histological changes of long standing and suggesting a prenatal or an early postnatal origin which

Fig . 9. Tissue rarefaction and spongy form changes in the subependymal layer. (Case 4, occipital hom) (HE x 200).

Fig. 10. Large reactive asrrocytes in the cerebral white mat ­ter showing intracytoplasmic vacuoles and cytopathological as­peel of lipidized cells. (Case 6. white matter of the centrum semiovale) (GFAP X 400).

may underlie CNS dysfunction invol ving cardiorespira­tory control.

Most of our findings are in keeping with a pathological process involving the ependyma in SillS. Nevertheless, none of the infants presented clinical signs/symptoms be­fore death and neither the eNS histopathological findings nor other findings were sufficient to explain death. The study and further characterization of these ependymal changes may well be of help in recognizing possible pathological processes which may alter the normal de­velopment of some CNS structures in a particularly vul­nerable period.

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Received May 15. 1995 Revisions received October 20. 1995 and November 10. 1995 Accepted December 5. 1955

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