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Ependymal abnormalities in cerebro-hepato-renal disease of Zellweger
Brain & Development, 15 (1993) 270-277 0387-7604/93/$06.00 ,~] 1993 Elsevier Science Publishers B.V. All rights reserved BRADEV 00073
Ependymal abnormalities in cerebro-hepato-renal disease of Zellweger H a r v e y B. S a r n a t a'bx, M D , F R C P C , C y n t h i a L. T r e v e n e n a, M D , F R C P C a n d H u s a m Z. D a r w i s h b'c M D , F R C P C
Departments of apathology, bPaediatrics and 'Clinical Neurosciences, University of Calgary Faculty of Medicine and Alberta Children's Hospital, Calgary, Alta, Canada Received 29 January 1993; accepted 21 April 1993 The ependyma in six infants dying with cerebro-hepato-renal (CHR) disease showed similar but less extensive morphological and immunohistochemical abnormalities to those previously demonstrated in lissencephaly/pachygyria. More than two-thirds of the ependyma lining all ventricles was a pseudostratified columnar epithelium resembling midfetal life. Discontinuities did not correlate with minimal ventriculomegaly. Subventricular rosettes were common and not confined to regions of gaps in the overlying ependyma. Subependymal gliosis and glial nodules were absent. Immunoreactivity of ependymal cells for vimentin and GFAP was normal for age. but abnormally positive for S-100 protein and cytokeratin, as in lissencephaly; unlike lissencephaly, the rosettes in CHR disease also are reactive. Ependymal abnormalities may contribute to the pathogenesis of cerebral dysgenesis in CHR disease as in other genetic disorders of neuroblast migration. Key words." Cerebro-hepato-renal disease; Ependyma; S-100 protein; Zellweger disease
INTRODUCTION Cerebro-hepato-renal (CHR) disease of Zellweger is an autosomal recessive trait expressed as malformations of multiple organ systems. Clinical abnormalities include intrahepatic biliary dysgenesis and multiple renal cortical cysts leading to liver and kidney failure, intrauterine growth retardation, developmental delay, muscular hypotonia, seizures and usually death in early infancy [1-5]. Metabolic defects consist of decreased or absent peroxisomes [6-8], defective pipecolic acid metabolism [7,9] deficient biosynthesis of plasmalogens [9,10] and abnormalities in mitochondrial structure and function [7,9,10]. The principal pathological lesions in the central nervous system are due to severe disturbances in neuroblast migration in the inferior olivary nuclei, cerebellum and cerebral hemispheres, glial nodules, neurofibrillary changes, delayed myelination of central pathways and abnormal formation of dendrites and synapses [11-16]. Cytoarchitectonic studies of the neocortex show that only some neurons of layers 2 and 3 are in their normal positions. The rest and those of all other layers are heterotopic [15]. The fetal ependyma is a heterogeneous, secretory Correspondence address: Dr. H.B. Sarnat, Children's Hospital and Medical Center, Neurology CH-49, 4800 Sand Point Way NE, Seattle, WA 98105, USA. Fax: (1) (206) 528 2649.
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structure that dynamically regulates several developmental processes of the maturing nervous system, including the arrest of mitotic activity in the neuroepithelium, the guidance of axonal growth cones and probably the maintenance and transformation of radial glial cells that guide migratory neuroblasts [17,18]. We have recently demonstrated extensive ependymal abnormalities in a genetically diverse group of cases of lissencephaly/pachygyria that includes type I (MillerDieker syndrome), type II (Fukuyama congenital muscular dystrophy; Walke~Warburg syndrome) and hemimegalencephalic pachygyria [19]. The present study was undertaken because CHR disease is another genetic disorder of neuroblast migration beginning early in gestation. The demonstration of similar morphological and metabolic abnormalities in infants with CHR disease would support the hypothesis that the fetal ependyma is a primary factor in pathogenesis. MATERIALS AND METHODS
Eight infants were diagnosed as CHR disease in the neonatal period at Foothills Provincial Hospital or Alberta Children's Hospital in Calgary between 1981 and 1992. All of these children died within the first 9 months and six were available for postmortem examination. Vastus lateralis muscle biopsies had been perBrain & Development, Vol 15, No 4, 1993
formed in four of the cases before death. All 6 infants in this study were born at term and none had experienced complications during the pregnancies or had shown evidence of fetal distress except Case 2 who suffered intrapartum asphyxia and meconium aspiration. Apgar scores were unexpectedly low in all cases, and three (Cases 1, 3 and 5) required intubation at birth; the others responded to oxygen by mask and gentle resuscitative efforts. Clinical features shared by all patients in this study were the typical facies of CHR disease, generalized muscular hypotonia and weakness, and global developmental delay. Seizures were observed in Cases 1, 4 and 5. Elevated serum and urinary pipecolic acid was demonstrated in all except Case 6. Case 5 also had a 10fold increase in urinary cystathionine and a 5-fold increase in serum serine and threonine; serum tyrosine was elevated in Case 3. Cases 2, 3 and 6 showed ranges of fibroblast plasmalogen concentrations and phosphate acyltransferase activity consistent with CHR disease; impaired plasmalogen biosynthesis also was demonstrated. Thorough gross and microscopic postmortem examinations were performed using established methods of pediatric pathology. The brain and spinal cord were examined in each case using standard neuropathological techniques that included representative sections stained with hematoxylin-eosin and hematoxylin-eosin/Luxol fast blue for myelin. The ependyma was systematically examined in all horns of the lateral ventricles, third and fourth ventricles, and spinal central canal at various levels. lmmunohistochemical studies included peroxidaseantiperoxidase stains for vimentin, glial fibrillary acidic protein (GFAP), S-100 protein and monoclonal antibodies for cytokeratin CK-904 (molecular weight 68
kDa). The methods employed were described previously [23]. Acridine orange fluorochrome stains were prepared and examined in the fluorescence microscope for ribonucleic acid (RNA) in the cytoplasm of ependymal cells. All immunohistochemical and Acridine orange studies were performed on formalin-fixed, paraffin-embedded sections. Comparisons were made with three normal term neonatal control brains previously described [17] and with normal infant brains at 4, 6 and 9 months of age. RESULTS
General neuropathological findings The gross and microscopic findings were similar in all cases and are summarized in Table I. The pattern of cerebral convolutions was abnormal in all cases. Coronal sections of the cerebral hemispheres revealed only minimal ventriculomegaly (Fig 1). Periventricular cysts or cavities were demonstrated over the heads of the caudate nuclei bilaterally in all cases except Case 5; no associated hemorrhages were identified. Microscopic examination of the cerebral hemispheres disclosed poorly laminated neocortex with many displaced and disoriented neurons. In some places, the architecture of the cerebral cortex resembled the four layers of classical lissencephaly, but in most places it was not as well organized or distinctive. Histological evidence of ischemic/hypoxic encephalopathy was absent or minimal except in Case 2 that showed extensive neuronal loss in the hippocampus, neocortex and among Purkinje cells of the cerebellum; surviving neurons in this case often showed chromatolysis, eosinophilic cytoplasm and other cytological changes consistent with a subacute hypoxic insult. None of the periventricular cysts were associated with fresh or old hemorrhage and no hemosiderin deposits
Table I
Summary of general neuropathological findings in cases of cerebro-hepato-renal ( CHR) disease Case
Sex
Age at death
Gestation- Birth al age at weight birth (g) (week)
Brain weight (g)
Abn. gyri
Abn. migr.
Cerebel. hamart,
Periventr. cysts
Hypomyel.
Other
M
3 days
40
3,330
436
+
+
-
+
+
F
3.5 months 40
3,115
612
+
+
-
+
+
M F M F
9 months 4 months 1 month 7 days
2,640 2,360 3,250 2,940
721 594 395 406
+ + + +
+ + + +
+ + + -
+ + +
+ + + +
Olf. bulb absent R, hypoplastic L; partial callosal agenesis Neuronal loss, hippocampus and cerebellum Many fused gyri Inf. olives small and poorly formed Large cavum vergae Many fused gyri
41 40 40 40
a b n , a b n o r m a l ; cerebel, cerebellar; gest, gestational; h a m a r t , h a m a r t o m a t a ; h y p o m y e l , h y p o m y e l i n a t i o n ; m i g r , m i g r a t i o n ; olf, olfactory; periventr, periventricular.
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Fig 1. Coronal gross sections of cerebral hemispheres of (A) Case I, (B) Case 2 and (C) Case 3. The lateral ventricles and third ventricle are only minimally dilated. A prominent cavum septi pellucidi is present. Periventricular cavities are seen (arrows) over the caudate nuclei, but are not associated with hemorrhages. The grey matter is thicker than expected for normal age-matched controls. Nodularity is not seen at the ependymal surfaces.
"
i,b
were demonstrated in the tissue. The cysts were not lined by ependyma.
Ependymal findings The ependymal abnormalities were similar in all cases and are described together. About two-thirds of the surface of the lateral, third and fourth ventricles was lined by pseudostratified columnar ependymal epithelium that was partially citiated (Fig 2). The remainder of the ependyma was a simple cuboidal ciliated epithelium. Multiple discontinuities of the ependyma were seen in the lateral ventricles involving the frontal, tern272
Fig 2. Ependyma lining the temporal horn of the lateral ventricle in (upper panel) Case 1 and (lower panel) Case 6 is a pseudostratified columnar epithelium reminiscent of a 30 week fetus. The undulation of the ventricular surface (upper panel) is associated with minor sulcation but no subependymal gliosis or glial nodule formation. A subventricular rosette (arrow) is not associated with discontinuity in the overlying ependyma at the ventricular surface. (Hematoxylin-eosin, x 42).
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Fig 3. Case 2. A discontinuity in the ependyma of the frontal horn over the thalamus is not accompanied by subventricular gliosis. At one end of the ependyma, a rosette is budding from the terminal surface ependyma (arrow). (Hematoxylin-eosin, x 42).
poral and occipital horns and, to a lesser extent, in the third and fourth ventricles. The gaps were most frequent in regions of subventricular cyst formation. Rosettes were sometimes seen budding from the terminal end of the ependymal epithelium (Fig 3). Ependymal rosettes also were found commonly in the subventricular region beneath continuous stretches of ependyma (Fig 4) as well as in regions of the subventricular zone not overlined by ependyma at the ventricular surface. Undulations and minor sulcation sometimes occurred in the ventricular surface (Fig 2). but deep sulcation of the ventricles was not found and subependymal nodules of proliferated glial cells and processes were conspicuously absent. The central canal of the spinal cord was generally formed by a pseudostratified columnar epithelium, but
Fig 4. Case 2. A line of ependymal rosettes is seen in the upper medulla oblongata beneath the floor of the fourth ventricle, lateral to the sulcus limitans. The ependyma at the ventricular surface is a pseudostratified columnar epithelium, but becomes a simple cuboidal epithelium and atrophic with a gap of one or two cells at the site of origin of the rosettes (arrow). (Hematoxylin-eosin, x 42).
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Fig 5. Case 2. The central canal of the cervical spinal cord is formed by a pseudostratified columnar epithelium that is deficient at the roof plate (rp). True rosettes (arrowheads) and pseudorosettes (arrows) are formed by ependymal cells in the zone surrounding the central canal. The true rosettes were strongly reactive for S-100 protein. (Hematoxylin-eosin, x 104).
discontinuities occurred similar to those of the lateral ventricles. Gaps in the central canal ependyma was generally in the region of the roof plate (Fig 5) but occasionally also involved the lateral wall of the central canal above the sulcus limitans, i.e. the alar plate. The
Fig 6. Case 6. Some ependymal cells in the lateral part of the floor of the fourth ventricle have fetal basal processes and are strongly reactive for G F A P (arrows); others are non-reactive. (GFAP, × 150).
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B A
Fig 7. Case 3. lmmunoreactivity for 5 100 protein (A) is strong in ependymal cells lining the temporal horn of the lateral ventricle, but (B) is weak though still present in the discontinuous ependyma of the occipital horn. Subependymal astrocytes (a) are strongly reactive and neurons (n) are nonreactive. C: Case 6. Ependymal rosettes beneath the lateral ventricle (arrows) are strongly immunoreactive for S-100 protein. The overlying ventricular surface (v) lacks an ependymal lining. ( x 250).
floor plate was not involved in any of our cases. Both true rosettes and pseudorosettes of ependymal cells were seen in association with these gaps (Fig 5) at cervical, lumbar and sacral levels. No mitotic figures were found in any region of the ependyma of the spinal cord or brain. Most ependymal cells were not immunoreactive for vimentin or for G F A P , but scattered cells, particularly those still possessing fetal basal processes, were reactive (Fig 6). These cells were generally limited to regions of the ependyma where such reactivity is normally expected in the immediate postnatal period [17], and they were not demonstrated in infants older than 3 months. The ependyma of all ventricles was reactive for S-100 protein at all ages including Case 3, a 9-month-old and the oldest in this study. Some regions showed stronger reactivity than others (Fig 7A,B), but no consistent pattern could be identified with respect to the pseudostratified or simple cuboidal architecture or to regional distribution. Many subventricular ependymal rosettes, including those around the ventricles (Fig 7C) and the spinal central canal, also were strongly reactive. Subependymal astrocytes were reactive for S-100 protein 274
and for G F A P , a normal finding. Weak immunoreactivity for cytokeratin CK-904 was demonstrated in most ependymal cells at the ventricular surfaces. Acridine orange R N A fluorescence was strong in all differentiated ependymal cells, regardless of organization as a pseudostratified or simple columnar epithelium or as rosettes in the subventricular zone. The control neonatal brains showed only minor gaps in the ependyma and occasional rosettes at the angles of the ventricles. Vimentin, G F A P and cytokeratin were expressed in less than 15 percent of ependymal cells of the lateral and third ventricles and were rare in the fourth; S-100 protein was expressed in just over half the ependymal cells lining the lateral ventricles and about 10-20% of cells lining the third and fourth ventricles. By 6 months of age immunoreactivity for any of the proteins studied was rare in ependymal cells in all sites. None of the occasional ependymal rosettes in normal neonatal or older brains expressed vimentin, G F A P , cytokeratin or S-100 protein.
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DISCUSSION The mechanism of arrested neuroblast migration in C H R disease remains elusive, but it is speculated that regional tissue constraints such as density of radial glia and the interweave of aberrant axons may play a role [16] or that circulating metabolites behave as neurotoxins to impede migration [15]. The decreased plasmalogens and increased very long-chain fatty acids resulting from the documented peroxisomal and mitochondrial abnormalities are uncertain factors in neuroblast migration. A possible role of the ependyma in pathogenesis has not previously been considered. The ependyma is a secretory structure that participates in several neuroembryological processes including neuroblast migration, probably by modulating the function and transformation of radial glial cells that guide migratory neuroblasts [18]. Fetal ependymal cells express proteins normally absent from mature ependymal cells: structural cytoskeletal proteins, such as vimentin and GFAP, and secretory molecules such as proteoglycans and S-100 protein [17,18,20,21]. The ependymal abnormalities here demonstrated in CHR disease are qualitatively similar but quantitatively less extensive than those found in classical lissencephaly/pachygyria of diverse etiologies [19]. Shared features of the ependyma in C H R disease and lissencephaly include a persistent pseudostratified columnar arrangement resembling the normal condition at midgestation, discontinuities disproportionate to the minor degree of ventricular dilatation, subventricular rosette formation, and persistence of high fetal concentrations of S-100 protein and high molecular weight cytokeratin, coupled with a normal loss of G F A P immunoreactivity expected for the age. Strong vimentin expression continues postnatally in the W a l k e ~ W a r burg syndrome but not in other forms of lissencephaly/pachygyria [19] or in our cases of C H R disease. The expression of S-100 protein in our cases of CHR disease qualitatively was stronger and involved more ependymal cells th~n in any of the controls. One difference between C H R disease and lissencephaly/pachygyria is the lack of subventricular gliosis and glial nodule formation in CHR; this absence may be related in part to the younger ages of the C H R population studied, all of whom were young infants because of early death as the natural history of the disease. Most of our lissencephaly cases were children as old as 7 years. The glial nodules may be chronic reactive changes that take months or years to develop, as with similar nodules that form following inflammatory conditions such as ependymitis (i.e. ventriculitis). Brain & Development, Vol 15, No 4, 1993
Five of our 6 cases of C H R disease had subventricular cysts over the heads of the caudate nuclei (Fig 1). as has been previously described in this disease [13]. These cysts were not associated with evidence of either recent or old hemorrhage or with other features to suggest periventricular leukomalacia (though our cases were born at term) or ischemic/hypoxic encephalopathy. Similar cysts are not usually found in lissencephaly or other neuroblast migratory disorders except in the Walker-Warburg syndrome. The pathogenesis of the cysts and their relation to the ependymal abnormalities are uncertain. They are in a site where radial glial cell bodies could be involved in necrosis. Clusters of subventricular rosettes of ependymal cells near the angles of the ventricles and mild sulcation of the cerebral aqueduct are normal developmental features [22-24] and it may be difficult at times to distinguish genuine pathological changes from minor variations of normal ontogenesis. Pseudorosettes, by contrast, are rare in normal brains and spinal cords. The pericapillary rosettes in the human spinal cord reported by Sasaki and Maruyama [25] are probably axonal swellings unrelated to the structures here described. Strong immunoreactivity for S-100 protein was demonstrated in the ependymal rosettes in C H R disease (Fig 7C), unlike the absence of S-100 reactivity in lissencephaly [19], suggesting that the rosettes remain metabolically active in C H R disease but become inert in classical lissencephaly or perhaps with aging. Discontinuities or gaps in the ependyma are consistently found in hydrocephalic brains, presumably from stretching. Quantitative data correlating ventriculomegaly with the extent of such gaps are not yet available (Sarnat, in preparation), but in our experience the ependymal discontinuities in C H R disease and in lissencephaly are greater than can be attributed to ependymal stretching alone (Fig 1) and may occur even in the absence of ventricular enlargement. The capacity of the mature ependyma to regenerate or repair itself is very limited. In experimental hydrocephalus in hamsters, the ependyma becomes discontinuous with progressive ventricular dilatation and the gaps become filled with astrocytic processes; remaining ependymal cells do not exhibit mitotic activity and subventricular glial cells do not undergo metaplasia to form new ependymal cells [26]. The transplantation of fetal cerebral cortical homografts into the spinal cord of adult rats is, however, associated with mitotic proliferation of new ependymal cells to line the cysts within the graft [27]. No ependymal proliferation was seen in our cases of CHR disease or lissencephaly. CHR disease is one of several peroxisomal disorders 275
that also include neonatal adrenoleukodystrophy and infantile Refsum disease; these diseases share some neuropathological features such as defective myelination [28-31]. Abnormal neuroblast migration is a component of autosomal recessive neonatal adrenoleukodystrophy as in C H R disease. The ependyma in these other peroxisomal diseases is not reported to be altered but may not have been examined critically; even previous neuropathological studies of C H R disease neglect the ependyma [12,13], as do most reports of lissencephaly. Peroxisomes are more numerous in immature than in mature nerve cells [32], but ependymal cells have not been examined ultrastructurally for peroxisomes during development, at maturity, or in C H R or other peroxisomal diseases. Whether the ependymal abnormalities are secondary and o f minor importance, as often is assumed, or whether they are primary and contribute to pathogenesis is incompletely resolved, but the proved role of the fetal ependyma in normal cerebral development and the abnormalities demonstrated in neuroblast migratory disorders strongly suggest an importance more than as a mere decorative lining of the ventricular system in the cerebral dysgenesis of C H R disease. ACKNOWLEDGEMENTS
Drs. N.B. Rewcastle, B. Curry, and G. Machin performed the primary neuropathological examinations in three of the cases. The plasmalogen studies in Case 3 were performed by Prof. R.B.H. Schutgens at the University of Amsterdam, the Netherlands; similar studies of Cases 2 and 6 were performed by Dr. H.W. Moser at the KennedyKrieger institute, Johns Hopkins University- Baltimore, Maryland, USA. Ms. P. Orton and Ms. L. Hines provided technical support in the histopathological preparations. This work was supported by Grant 86950-0898 to H.B.S. from the Alberta Children's Hospital Foundation.
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cord. Acta Neuropathol 1992; 83: 598- 604. 26. Collins P, Fairman S. Repair of the ependyma in hydrocephalic brains. Neuropathol Appl Neurobiol 1990; 16: 45-56. 27. Bernstein JJ. Ependymal formation in adult rat spinal cord after transplantation of fetal cerebral cortex homografts. J Neurosci Res 1986; 15: 481- 90. 28. Poll-The BT, Saudubray JM, Ogier HA, et al. Infantile Refsum disease an inherited peroxisomal disorder: comparison with Zellweger syndrome and neonatal adrenoleukodystrophy. Eur J Pediatr 1987; 146: 477- 83. 29. Sarda H, Henry V, Le Loc'h H, Aubourg P, Poll-The BT, Saudubray JM. Adrenoleukodystrophie n6o-natale. A p r o p o s de trois
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cas d'une m~me fratrie. Arch Fr Pediatr (Paris) 1989; 36:233 236. 30. Barth PG, Schutgens RBH, Wanders RJ, Heymans HSA. The spectrum of peroxisomal disorders. In: French JH, Harel S, Casaer P, eds. ChiM neurology and development disabilities. Baltimore: Paul H Brookes Publ Co, 1989:67 82. 31. Moser HW. Peroxisomes and pediatric neurological diseases. In: Fukuyama Y, Suzuki Y, Kamoshita S, Casaer P, eds. Fetal and perinatal neurology Basel: Karger, 1992: 268-284. 32. Holtzman E. Peroxisomes in nervous tissue. Ann N Y Acad Sci 1982; 386: 523-525.
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Ependymal abnormalities in cerebro-hepato-renal disease of Zellweger H a r v e y B. S a r n a t a'bx, M D , F R C P C , C y n t h i a L. T r e v e n e n a, M D , F R C P C a n d H u s a m Z. D a r w i s h b'c M D , F R C P C
Departments of apathology, bPaediatrics and 'Clinical Neurosciences, University of Calgary Faculty of Medicine and Alberta Children's Hospital, Calgary, Alta, Canada Received 29 January 1993; accepted 21 April 1993 The ependyma in six infants dying with cerebro-hepato-renal (CHR) disease showed similar but less extensive morphological and immunohistochemical abnormalities to those previously demonstrated in lissencephaly/pachygyria. More than two-thirds of the ependyma lining all ventricles was a pseudostratified columnar epithelium resembling midfetal life. Discontinuities did not correlate with minimal ventriculomegaly. Subventricular rosettes were common and not confined to regions of gaps in the overlying ependyma. Subependymal gliosis and glial nodules were absent. Immunoreactivity of ependymal cells for vimentin and GFAP was normal for age. but abnormally positive for S-100 protein and cytokeratin, as in lissencephaly; unlike lissencephaly, the rosettes in CHR disease also are reactive. Ependymal abnormalities may contribute to the pathogenesis of cerebral dysgenesis in CHR disease as in other genetic disorders of neuroblast migration. Key words." Cerebro-hepato-renal disease; Ependyma; S-100 protein; Zellweger disease
INTRODUCTION Cerebro-hepato-renal (CHR) disease of Zellweger is an autosomal recessive trait expressed as malformations of multiple organ systems. Clinical abnormalities include intrahepatic biliary dysgenesis and multiple renal cortical cysts leading to liver and kidney failure, intrauterine growth retardation, developmental delay, muscular hypotonia, seizures and usually death in early infancy [1-5]. Metabolic defects consist of decreased or absent peroxisomes [6-8], defective pipecolic acid metabolism [7,9] deficient biosynthesis of plasmalogens [9,10] and abnormalities in mitochondrial structure and function [7,9,10]. The principal pathological lesions in the central nervous system are due to severe disturbances in neuroblast migration in the inferior olivary nuclei, cerebellum and cerebral hemispheres, glial nodules, neurofibrillary changes, delayed myelination of central pathways and abnormal formation of dendrites and synapses [11-16]. Cytoarchitectonic studies of the neocortex show that only some neurons of layers 2 and 3 are in their normal positions. The rest and those of all other layers are heterotopic [15]. The fetal ependyma is a heterogeneous, secretory Correspondence address: Dr. H.B. Sarnat, Children's Hospital and Medical Center, Neurology CH-49, 4800 Sand Point Way NE, Seattle, WA 98105, USA. Fax: (1) (206) 528 2649.
270
structure that dynamically regulates several developmental processes of the maturing nervous system, including the arrest of mitotic activity in the neuroepithelium, the guidance of axonal growth cones and probably the maintenance and transformation of radial glial cells that guide migratory neuroblasts [17,18]. We have recently demonstrated extensive ependymal abnormalities in a genetically diverse group of cases of lissencephaly/pachygyria that includes type I (MillerDieker syndrome), type II (Fukuyama congenital muscular dystrophy; Walke~Warburg syndrome) and hemimegalencephalic pachygyria [19]. The present study was undertaken because CHR disease is another genetic disorder of neuroblast migration beginning early in gestation. The demonstration of similar morphological and metabolic abnormalities in infants with CHR disease would support the hypothesis that the fetal ependyma is a primary factor in pathogenesis. MATERIALS AND METHODS
Eight infants were diagnosed as CHR disease in the neonatal period at Foothills Provincial Hospital or Alberta Children's Hospital in Calgary between 1981 and 1992. All of these children died within the first 9 months and six were available for postmortem examination. Vastus lateralis muscle biopsies had been perBrain & Development, Vol 15, No 4, 1993
formed in four of the cases before death. All 6 infants in this study were born at term and none had experienced complications during the pregnancies or had shown evidence of fetal distress except Case 2 who suffered intrapartum asphyxia and meconium aspiration. Apgar scores were unexpectedly low in all cases, and three (Cases 1, 3 and 5) required intubation at birth; the others responded to oxygen by mask and gentle resuscitative efforts. Clinical features shared by all patients in this study were the typical facies of CHR disease, generalized muscular hypotonia and weakness, and global developmental delay. Seizures were observed in Cases 1, 4 and 5. Elevated serum and urinary pipecolic acid was demonstrated in all except Case 6. Case 5 also had a 10fold increase in urinary cystathionine and a 5-fold increase in serum serine and threonine; serum tyrosine was elevated in Case 3. Cases 2, 3 and 6 showed ranges of fibroblast plasmalogen concentrations and phosphate acyltransferase activity consistent with CHR disease; impaired plasmalogen biosynthesis also was demonstrated. Thorough gross and microscopic postmortem examinations were performed using established methods of pediatric pathology. The brain and spinal cord were examined in each case using standard neuropathological techniques that included representative sections stained with hematoxylin-eosin and hematoxylin-eosin/Luxol fast blue for myelin. The ependyma was systematically examined in all horns of the lateral ventricles, third and fourth ventricles, and spinal central canal at various levels. lmmunohistochemical studies included peroxidaseantiperoxidase stains for vimentin, glial fibrillary acidic protein (GFAP), S-100 protein and monoclonal antibodies for cytokeratin CK-904 (molecular weight 68
kDa). The methods employed were described previously [23]. Acridine orange fluorochrome stains were prepared and examined in the fluorescence microscope for ribonucleic acid (RNA) in the cytoplasm of ependymal cells. All immunohistochemical and Acridine orange studies were performed on formalin-fixed, paraffin-embedded sections. Comparisons were made with three normal term neonatal control brains previously described [17] and with normal infant brains at 4, 6 and 9 months of age. RESULTS
General neuropathological findings The gross and microscopic findings were similar in all cases and are summarized in Table I. The pattern of cerebral convolutions was abnormal in all cases. Coronal sections of the cerebral hemispheres revealed only minimal ventriculomegaly (Fig 1). Periventricular cysts or cavities were demonstrated over the heads of the caudate nuclei bilaterally in all cases except Case 5; no associated hemorrhages were identified. Microscopic examination of the cerebral hemispheres disclosed poorly laminated neocortex with many displaced and disoriented neurons. In some places, the architecture of the cerebral cortex resembled the four layers of classical lissencephaly, but in most places it was not as well organized or distinctive. Histological evidence of ischemic/hypoxic encephalopathy was absent or minimal except in Case 2 that showed extensive neuronal loss in the hippocampus, neocortex and among Purkinje cells of the cerebellum; surviving neurons in this case often showed chromatolysis, eosinophilic cytoplasm and other cytological changes consistent with a subacute hypoxic insult. None of the periventricular cysts were associated with fresh or old hemorrhage and no hemosiderin deposits
Table I
Summary of general neuropathological findings in cases of cerebro-hepato-renal ( CHR) disease Case
Sex
Age at death
Gestation- Birth al age at weight birth (g) (week)
Brain weight (g)
Abn. gyri
Abn. migr.
Cerebel. hamart,
Periventr. cysts
Hypomyel.
Other
M
3 days
40
3,330
436
+
+
-
+
+
F
3.5 months 40
3,115
612
+
+
-
+
+
M F M F
9 months 4 months 1 month 7 days
2,640 2,360 3,250 2,940
721 594 395 406
+ + + +
+ + + +
+ + + -
+ + +
+ + + +
Olf. bulb absent R, hypoplastic L; partial callosal agenesis Neuronal loss, hippocampus and cerebellum Many fused gyri Inf. olives small and poorly formed Large cavum vergae Many fused gyri
41 40 40 40
a b n , a b n o r m a l ; cerebel, cerebellar; gest, gestational; h a m a r t , h a m a r t o m a t a ; h y p o m y e l , h y p o m y e l i n a t i o n ; m i g r , m i g r a t i o n ; olf, olfactory; periventr, periventricular.
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271
Fig 1. Coronal gross sections of cerebral hemispheres of (A) Case I, (B) Case 2 and (C) Case 3. The lateral ventricles and third ventricle are only minimally dilated. A prominent cavum septi pellucidi is present. Periventricular cavities are seen (arrows) over the caudate nuclei, but are not associated with hemorrhages. The grey matter is thicker than expected for normal age-matched controls. Nodularity is not seen at the ependymal surfaces.
"
i,b
were demonstrated in the tissue. The cysts were not lined by ependyma.
Ependymal findings The ependymal abnormalities were similar in all cases and are described together. About two-thirds of the surface of the lateral, third and fourth ventricles was lined by pseudostratified columnar ependymal epithelium that was partially citiated (Fig 2). The remainder of the ependyma was a simple cuboidal ciliated epithelium. Multiple discontinuities of the ependyma were seen in the lateral ventricles involving the frontal, tern272
Fig 2. Ependyma lining the temporal horn of the lateral ventricle in (upper panel) Case 1 and (lower panel) Case 6 is a pseudostratified columnar epithelium reminiscent of a 30 week fetus. The undulation of the ventricular surface (upper panel) is associated with minor sulcation but no subependymal gliosis or glial nodule formation. A subventricular rosette (arrow) is not associated with discontinuity in the overlying ependyma at the ventricular surface. (Hematoxylin-eosin, x 42).
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Fig 3. Case 2. A discontinuity in the ependyma of the frontal horn over the thalamus is not accompanied by subventricular gliosis. At one end of the ependyma, a rosette is budding from the terminal surface ependyma (arrow). (Hematoxylin-eosin, x 42).
poral and occipital horns and, to a lesser extent, in the third and fourth ventricles. The gaps were most frequent in regions of subventricular cyst formation. Rosettes were sometimes seen budding from the terminal end of the ependymal epithelium (Fig 3). Ependymal rosettes also were found commonly in the subventricular region beneath continuous stretches of ependyma (Fig 4) as well as in regions of the subventricular zone not overlined by ependyma at the ventricular surface. Undulations and minor sulcation sometimes occurred in the ventricular surface (Fig 2). but deep sulcation of the ventricles was not found and subependymal nodules of proliferated glial cells and processes were conspicuously absent. The central canal of the spinal cord was generally formed by a pseudostratified columnar epithelium, but
Fig 4. Case 2. A line of ependymal rosettes is seen in the upper medulla oblongata beneath the floor of the fourth ventricle, lateral to the sulcus limitans. The ependyma at the ventricular surface is a pseudostratified columnar epithelium, but becomes a simple cuboidal epithelium and atrophic with a gap of one or two cells at the site of origin of the rosettes (arrow). (Hematoxylin-eosin, x 42).
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Fig 5. Case 2. The central canal of the cervical spinal cord is formed by a pseudostratified columnar epithelium that is deficient at the roof plate (rp). True rosettes (arrowheads) and pseudorosettes (arrows) are formed by ependymal cells in the zone surrounding the central canal. The true rosettes were strongly reactive for S-100 protein. (Hematoxylin-eosin, x 104).
discontinuities occurred similar to those of the lateral ventricles. Gaps in the central canal ependyma was generally in the region of the roof plate (Fig 5) but occasionally also involved the lateral wall of the central canal above the sulcus limitans, i.e. the alar plate. The
Fig 6. Case 6. Some ependymal cells in the lateral part of the floor of the fourth ventricle have fetal basal processes and are strongly reactive for G F A P (arrows); others are non-reactive. (GFAP, × 150).
273
B A
Fig 7. Case 3. lmmunoreactivity for 5 100 protein (A) is strong in ependymal cells lining the temporal horn of the lateral ventricle, but (B) is weak though still present in the discontinuous ependyma of the occipital horn. Subependymal astrocytes (a) are strongly reactive and neurons (n) are nonreactive. C: Case 6. Ependymal rosettes beneath the lateral ventricle (arrows) are strongly immunoreactive for S-100 protein. The overlying ventricular surface (v) lacks an ependymal lining. ( x 250).
floor plate was not involved in any of our cases. Both true rosettes and pseudorosettes of ependymal cells were seen in association with these gaps (Fig 5) at cervical, lumbar and sacral levels. No mitotic figures were found in any region of the ependyma of the spinal cord or brain. Most ependymal cells were not immunoreactive for vimentin or for G F A P , but scattered cells, particularly those still possessing fetal basal processes, were reactive (Fig 6). These cells were generally limited to regions of the ependyma where such reactivity is normally expected in the immediate postnatal period [17], and they were not demonstrated in infants older than 3 months. The ependyma of all ventricles was reactive for S-100 protein at all ages including Case 3, a 9-month-old and the oldest in this study. Some regions showed stronger reactivity than others (Fig 7A,B), but no consistent pattern could be identified with respect to the pseudostratified or simple cuboidal architecture or to regional distribution. Many subventricular ependymal rosettes, including those around the ventricles (Fig 7C) and the spinal central canal, also were strongly reactive. Subependymal astrocytes were reactive for S-100 protein 274
and for G F A P , a normal finding. Weak immunoreactivity for cytokeratin CK-904 was demonstrated in most ependymal cells at the ventricular surfaces. Acridine orange R N A fluorescence was strong in all differentiated ependymal cells, regardless of organization as a pseudostratified or simple columnar epithelium or as rosettes in the subventricular zone. The control neonatal brains showed only minor gaps in the ependyma and occasional rosettes at the angles of the ventricles. Vimentin, G F A P and cytokeratin were expressed in less than 15 percent of ependymal cells of the lateral and third ventricles and were rare in the fourth; S-100 protein was expressed in just over half the ependymal cells lining the lateral ventricles and about 10-20% of cells lining the third and fourth ventricles. By 6 months of age immunoreactivity for any of the proteins studied was rare in ependymal cells in all sites. None of the occasional ependymal rosettes in normal neonatal or older brains expressed vimentin, G F A P , cytokeratin or S-100 protein.
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DISCUSSION The mechanism of arrested neuroblast migration in C H R disease remains elusive, but it is speculated that regional tissue constraints such as density of radial glia and the interweave of aberrant axons may play a role [16] or that circulating metabolites behave as neurotoxins to impede migration [15]. The decreased plasmalogens and increased very long-chain fatty acids resulting from the documented peroxisomal and mitochondrial abnormalities are uncertain factors in neuroblast migration. A possible role of the ependyma in pathogenesis has not previously been considered. The ependyma is a secretory structure that participates in several neuroembryological processes including neuroblast migration, probably by modulating the function and transformation of radial glial cells that guide migratory neuroblasts [18]. Fetal ependymal cells express proteins normally absent from mature ependymal cells: structural cytoskeletal proteins, such as vimentin and GFAP, and secretory molecules such as proteoglycans and S-100 protein [17,18,20,21]. The ependymal abnormalities here demonstrated in CHR disease are qualitatively similar but quantitatively less extensive than those found in classical lissencephaly/pachygyria of diverse etiologies [19]. Shared features of the ependyma in C H R disease and lissencephaly include a persistent pseudostratified columnar arrangement resembling the normal condition at midgestation, discontinuities disproportionate to the minor degree of ventricular dilatation, subventricular rosette formation, and persistence of high fetal concentrations of S-100 protein and high molecular weight cytokeratin, coupled with a normal loss of G F A P immunoreactivity expected for the age. Strong vimentin expression continues postnatally in the W a l k e ~ W a r burg syndrome but not in other forms of lissencephaly/pachygyria [19] or in our cases of C H R disease. The expression of S-100 protein in our cases of CHR disease qualitatively was stronger and involved more ependymal cells th~n in any of the controls. One difference between C H R disease and lissencephaly/pachygyria is the lack of subventricular gliosis and glial nodule formation in CHR; this absence may be related in part to the younger ages of the C H R population studied, all of whom were young infants because of early death as the natural history of the disease. Most of our lissencephaly cases were children as old as 7 years. The glial nodules may be chronic reactive changes that take months or years to develop, as with similar nodules that form following inflammatory conditions such as ependymitis (i.e. ventriculitis). Brain & Development, Vol 15, No 4, 1993
Five of our 6 cases of C H R disease had subventricular cysts over the heads of the caudate nuclei (Fig 1). as has been previously described in this disease [13]. These cysts were not associated with evidence of either recent or old hemorrhage or with other features to suggest periventricular leukomalacia (though our cases were born at term) or ischemic/hypoxic encephalopathy. Similar cysts are not usually found in lissencephaly or other neuroblast migratory disorders except in the Walker-Warburg syndrome. The pathogenesis of the cysts and their relation to the ependymal abnormalities are uncertain. They are in a site where radial glial cell bodies could be involved in necrosis. Clusters of subventricular rosettes of ependymal cells near the angles of the ventricles and mild sulcation of the cerebral aqueduct are normal developmental features [22-24] and it may be difficult at times to distinguish genuine pathological changes from minor variations of normal ontogenesis. Pseudorosettes, by contrast, are rare in normal brains and spinal cords. The pericapillary rosettes in the human spinal cord reported by Sasaki and Maruyama [25] are probably axonal swellings unrelated to the structures here described. Strong immunoreactivity for S-100 protein was demonstrated in the ependymal rosettes in C H R disease (Fig 7C), unlike the absence of S-100 reactivity in lissencephaly [19], suggesting that the rosettes remain metabolically active in C H R disease but become inert in classical lissencephaly or perhaps with aging. Discontinuities or gaps in the ependyma are consistently found in hydrocephalic brains, presumably from stretching. Quantitative data correlating ventriculomegaly with the extent of such gaps are not yet available (Sarnat, in preparation), but in our experience the ependymal discontinuities in C H R disease and in lissencephaly are greater than can be attributed to ependymal stretching alone (Fig 1) and may occur even in the absence of ventricular enlargement. The capacity of the mature ependyma to regenerate or repair itself is very limited. In experimental hydrocephalus in hamsters, the ependyma becomes discontinuous with progressive ventricular dilatation and the gaps become filled with astrocytic processes; remaining ependymal cells do not exhibit mitotic activity and subventricular glial cells do not undergo metaplasia to form new ependymal cells [26]. The transplantation of fetal cerebral cortical homografts into the spinal cord of adult rats is, however, associated with mitotic proliferation of new ependymal cells to line the cysts within the graft [27]. No ependymal proliferation was seen in our cases of CHR disease or lissencephaly. CHR disease is one of several peroxisomal disorders 275
that also include neonatal adrenoleukodystrophy and infantile Refsum disease; these diseases share some neuropathological features such as defective myelination [28-31]. Abnormal neuroblast migration is a component of autosomal recessive neonatal adrenoleukodystrophy as in C H R disease. The ependyma in these other peroxisomal diseases is not reported to be altered but may not have been examined critically; even previous neuropathological studies of C H R disease neglect the ependyma [12,13], as do most reports of lissencephaly. Peroxisomes are more numerous in immature than in mature nerve cells [32], but ependymal cells have not been examined ultrastructurally for peroxisomes during development, at maturity, or in C H R or other peroxisomal diseases. Whether the ependymal abnormalities are secondary and o f minor importance, as often is assumed, or whether they are primary and contribute to pathogenesis is incompletely resolved, but the proved role of the fetal ependyma in normal cerebral development and the abnormalities demonstrated in neuroblast migratory disorders strongly suggest an importance more than as a mere decorative lining of the ventricular system in the cerebral dysgenesis of C H R disease. ACKNOWLEDGEMENTS
Drs. N.B. Rewcastle, B. Curry, and G. Machin performed the primary neuropathological examinations in three of the cases. The plasmalogen studies in Case 3 were performed by Prof. R.B.H. Schutgens at the University of Amsterdam, the Netherlands; similar studies of Cases 2 and 6 were performed by Dr. H.W. Moser at the KennedyKrieger institute, Johns Hopkins University- Baltimore, Maryland, USA. Ms. P. Orton and Ms. L. Hines provided technical support in the histopathological preparations. This work was supported by Grant 86950-0898 to H.B.S. from the Alberta Children's Hospital Foundation.
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