Prognostic indicators for vision and mortality in shaken baby syndrome.

OBJECTIVE: To study ocular and nonocular signs of patients diagnosed as having "shaken baby syndrome" and determine prognostic indicators for vision and mortality. METHODS: Medical records of child abuse cases involving bilateral retinal hemorrhages were reviewed. Particular attention was paid to visual function and pupillary light reaction at the time of admission as well as the location of retinal hemorrhages, neuroimaging findings, ventilatory requirement, and associated skeletal injuries. These findings were correlated with visual prognosis and mortality. RESULTS: Thirty consecutive cases met the criteria for review. At the initial visit, mean age of the children was 9.3 months (range, 1-39 months) and 12 children (40%) had at least fix-and-follow vision. Preretinal and intraretinal hemorrhages (93% [n = 28] and 100% [n = 30]) were more common than vitreous hemorrhage (10% [n = 3]). Subdural hematomas were detected in 21 patients (70%). Twenty children (67%) had seizures and 16 (53%) required ventilatory support; bruises and long bone fractures were seen in 14 (47%) and 4 (13%) children, respectively. Eight patients died. All patients with nonreactive pupils on presentation died, while all patients with a pupillary light reaction lived (P<.001). Six (86%) of 7 patients with midline shift died, whereas 21 (91%) of 23 with no midline shift lived (P<.001). At follow-up, retinal hemorrhages had resolved in nearly all children by 4 months, and 16 children (73%) had at least fix-and-follow vision. Ventilatory requirement was associated with poorer vision (P<.01). CONCLUSIONS: Nonreactive pupils and midline shift of the brain structures correlate highly with mortality. Ventilatory requirement, but not visual acuity on presentation, predicts visual outcome.

Developmentally regulated neural protein EAP-300 is expressed by myocardium and cardiac neural crest during chick embryogenesis.

The spatiotemporal distribution of EAP-300 (embryonic avian polypeptide of 300 kDa) was analyzed in embryonic chick heart using immunohistochemistry and confocal microscopy. EAP-300 is a developmentally regulated protein initially characterized in neural cells from chick retina. Myocardial cells all along the early tubular heart were ubiquitously immunolabeled for EAP-300 by embryonic day 2 (E2, Stage 13)). At E5 (Stage 24), myocardial EAP-300 expression levels remained significant in both atrial and ventricular myocardium. At E6 (Stage 28), distinct populations of EAP-300 immunolabeled cells were also observed external to the heart, in septal mesenchymal tissue and neural ganglia adjacent to the outflow tract; these cell populations were confirmed as neural crest-derived by co-localization of EAP-300 and HNK-1. At E13 (Stage 39), myocardial immunolabeling for EAP-300 was no longer ubiquitous, but increasingly restricted to conduction tissues, including the atrioventricular bundle and subendocardial Purkinje cells. This restriction of immunolabeling could be demonstrated definitively at E15 (stage 41), by which stage subendocardial and periarterial Purkinje fibers were clearly immunoreactive for EAP-300 and several known markers of chick conduction tissue, including specific myosin heavy chain isoforms and connexin42, a gap junctional protein preferentially expressed by Purkinje fibers. Just prior to hatching at E21 (Stage 46), immunolabeling of conduction tissues was reduced, although still above that of non-conductile myocardium. This spatiotemporal map of cardiac EAP-300 expression indicates that it is independently and transiently expressed in early myocardium, cardiac conduction tissue, and neural crest derivatives during development.

Molecular characterization of EAP-300: a high molecular weight, embryonic polypeptide containing an amino acid repeat comprised of multiple leucine-zipper motifs.

In this study we report the biochemical and initial molecular characterization of EAP-300, a developmentally regulated embryonal protein that has been shown previously to be expressed by radial glia in various regions of the CNS, including putative glial barriers. In the present study we have shown that the 300 kDa EAP-300 polypeptide is developmentally regulated in all tissues expressing the protein, which include various PNS and CNS tissues and muscle. In neural tissue the protein is readily detected during early embryogenesis, subsequently down-regulated at later stages, and is not detected in the adult. In contrast to neural tissue, small amounts of the protein are expressed in heart, consistent with earlier studies which showed that EAP-300 expression was maintained in the Purkinje cells of the heart conduction system. Metabolic labeling demonstrates that EAP-300 is a phosphoprotein, and is fatty acylated based on incorporation of [3H]palmitate. We also show that the normal developmental down-regulation of EAP-300 by glia does not occur in vitro, and these data therefore suggest that the signal(s) that regulates EAP-300 gene expression during development in vivo is absent in dissociated cell cultures. We have also initiated molecular studies of EAP-300 by screening embryonic brain cDNA expression libraries with a mixture of EAP-300 monoclonal antibodies. Sequence analysis of partial EAP-300 cDNAs indicate that the protein is related, if not identical, to IFAPa-400, a developmentally regulated intermediate filament-associated protein in chick that is proposed to participate in cell differentiation. These studies also indicate that EAP-300 mRNA is developmentally regulated and is expressed by glial cells in putative CNS barrier structures. Our studies also suggest that two pools of EAP-300 may exist in cells, implying that unlike IFAPa-400 the EAP-300 protein may not always be associated with intermediate filaments. Interestingly, our studies demonstrate that EAP-300 contains a novel repeat amino acid domain comprised of multiple leucine-zipper motifs, which may contribute to its function during glial differentiation.

Expression of the barrier-associated proteins EAP-300 and claustrin in the developing central nervous system.

Immunohistochemistry of embryonic chick central nervous system (CNS) and immunocytochemistry of retinal cells were performed to compare and map the expression of two barrier-associated molecules. EAP-300 (embryonic avian polypeptide of 300 kDa) and claustrin (a 320 kDa extracellular matrix keratan sulfate proteoglycan) were both transiently expressed in CNS regions that are considered non-permissive to either neuron migration or axon growth. In the developing spinal cord, EAP-300 and claustrin were both expressed by the marginal zone early in development and by the roof plate later in embryogenesis. In the developing rhombencephalon, immunoreactivity for both molecules was also observed first in the marginal zone, and later expression was restricted mostly to the midline. In the mesencephalon, EAP-300 and claustrin were also localized to the midline, and this expression represented a continuation of the expression observed in the spinal cord roof plate and hindbrain ventral midline. In the developing retina and cerebellum, EAP-300 and claustrin were differentially expressed. In retina, EAP-300 and claustrin were expressed by Müller cells and the optic fiber layer, respectively. In cerebellum at embryonic day 12 (E12), EAP-300 was expressed by Bergman glia, but claustrin was not expressed until E15. Immunocytochemical staining of retinal and cerebellar cultures indicated that EAP-300 was expressed by a subset of radial astrocytes, as confirmed by double labeling experiments with a specific marker for radial astrocytes. These data indicate that in the absence of claustrin expression, EAP-300 was expressed specifically by radial astrocytes during developmental periods of neuron migration. Also, the coexpression of EAP-300 and claustrin in CNS regions considered to be non-permissive to neurite extension suggests that these two developmentally regulated proteins may be associated with barrier function in the developing CNS.

Distribution of the novel developmentally-regulated protein EAP-300 in the embryonic chick nervous system.

In a previous study we described a 300 kDa, developmentally regulated protein identified in embryonic chick neural retina with a monoclonal antibody. Because this protein has been shown to be undetectable in the adult nervous system, and the monoclonal antibody is species-specific, the protein has been named embryonal avian polypeptide of 300 kDa (EAP-300). In the present study we have analyzed the histological expression of EAP-300 during chick embryogenesis. In the developing nervous system, EAP-300 expression was detected as early as Stage 5 (19 h), and was subsequently down-regulated to undetectable levels in the adult. Of particular interest was the association of EAP-300 with putative barriers of neuronal growth, such as the telencephalon/diencephalon glial knot, the dorsal midline in the mesencephalon and the midline in myelencephalon, and the spinal cord roof plate. EAP-300 was also shown to be expressed by Bergmann glia during the period of granule cell migration in the cerebellum. The expression of EAP-300 by radial astrocytes was confirmed in culture by immunofluorescent co-labeling with a MAb to EAP-300 and the R5 MAb, which is a radial astrocyte-specific marker. It has also been shown that EAP-300, when immunopurified from embryonic brain under non-dissociating conditions, co-purifies with a neural keratan sulfate proteoglycan that is also associated with CNS barrier structures during brain development. The restricted expression of EAP-300 in nervous tissue, particularly in CNS barrier structures, suggests that EAP-300 may play an important, but transient, role in the development of the chick nervous system.

Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth.

Monoclonal antibodies have been used to identify a 320 kd keratan sulfate proteoglycan that is primarily expressed in the embryonic chick nervous system. Immunohistochemical localization of the proteoglycan shows that it is expressed by putative midline barrier structures in the developing chick central nervous system. When added to laminin or neural cell adhesion molecule that has been adsorbed onto nitrocellulose-coated dishes, the proteoglycan abolishes cell attachment and neurite outgrowth on these adhesive substrata. This effect can be reversed by keratanase treatment and incubation with a monoclonal antibody that recognizes the keratan sulfate chains of the proteoglycan. These data suggest that this neural keratan sulfate proteoglycan plays an important role in the modulation of neuronal cell adhesion during embryonic brain development.