Winnie W. Y. Li

Calpain and N-methyl-d-aspartate (NMDA)-induced excitotoxicity in rat retinas.

Calpain-mediated proteolysis has been implicated as a major process in neuronal cell death in both acute insults and the chronic neurodegenerative disorders in the central nerves system. However, activation of calpain also plays a protective function in the early phase of excitotoxic neuronal death. The exact role of calpains in neuronal death and recovery after exposure to N-methyl-D-aspartate (NMDA) is not clearly known. The purpose of present study was to examine the involvement of mu- and m-calpain in NMDA-induced excitotoxicity in the adult rat retina. Increased immunoreactivity of mu-calpain was noted in RGC layer cells and in the inner nuclear layer with maximal expression at 12 h after NMDA injection. This was further confirmed with Western blotting. TdT-mediated biotin-dUTP nick end labeling (TUNEL) positive cells in the inner retina co-localized with moderate or intense mu-calpain immunoreactivity. In contrast, there was no remarkable change in m-calpain immunoreactivity at any time point after NMDA injection. Simultaneous injection of 2 nmol of a calpain inhibitor (calpain inhibitor II) significantly reduced the number of TUNEL-positive cells in the inner retina at 18 h after NMDA injection and preserved RGC-like cells counted at 7 days after injection. The results of this study showed that mu-calpain may be involved in mediating NMDA-induced excitotoxicity in the rat retina and calpain inhibitors may play a therapeutic role in NMDA related disease.

Proliferation and apoptosis in the epithelium of the developing human cornea and conjunctiva

To determine the distribution of proliferating and apoptotic cells in the human cornea during prenatal and early postnatal development, we examined sections of the bulbar conjunctiva, the limbus as well as the central and peripheral cornea between 11 weeks of gestation and 6 months after birth. The objective was to localize dividing cells by proliferating cell nuclear antigen-like immunoreactivity (PCNA-LI) and apoptotic cells by terminal transferase-mediated nick-end labeling (TUNEL). Before the 17th gestational week, PCNA-LI was absent in all 4 regions examined, indicating negligible cell proliferation during early development. After 20 weeks, strong PCNA-labeling was observed in all regions examined suggestive of high proliferative activity not only in the limbus and the bulbar conjunctiva, but also in the central and peripheral cornea. This rise in proliferative activity was followed by a steady decline: after 28 weeks, anti-PCNA staining gradually disappeared in the central and peripheral cornea, so that, at 6 months after birth, it was confined to the limbus and the bulbar conjunctiva, resembling the picture described for the adult cornea. TUNEL-positive cells were virtually absent in all 4 regions examined before the 38th gestational week. Apoptotic cells only started to appear at 38 weeks; at this stage, they were confined to the bulbar conjunctival epithelium. At 6 months after birth, TUNEL-positive cells were observed in the bulbar conjunctival epithelium and the entire cornea; the limbus, however remained devoid of apoptotic cells throughout the entire prenatal and early postnatal period. The present study for the first time localizes proliferating and apoptotic cells in the epithelium of the developing human cornea. Three stages of development can be distinguished: Minimal proliferation (until 17th week), vigorous proliferation over the entire cornea including the limbus and the bulbar conjunctiva (until 28th week) and gradual decrease in proliferative activity (after 28th week) accompanied by the appearance of apoptotic cells.

Axonal sprouting in the hemisected adult rat spinal cord

The morphological and biochemical changes were studied in adult Sprague-Dawley rats after hemisection at the L3 spinal cord level. After survival periods of one, two and three months, fluorescent tracers, FluoroGold or rhodamine B, were implanted into the dorsal white columns of these rats at the positions of the corticospinal tract below the lesion. Following uptake of the tracer, the rats were killed and the motor cortices and spinal cords of both control and hemisected rats were analysed for positively labelled neurons. The highest number of labelled cells were found two months after hemisection. They were present in both sides of the cortices, particularly in the contralateral cortex, and also in the gray matter of the spinal cord above the hemisection. A few rats which were subjected to complete transection of the spinal cord also showed labelling of neurons in the motor cortex two months after lesion. The Protargol silver technique and the [3H]choline uptake study confirmed the presence of nerve fibres traversing the lesion site in the hemisected spinal cord. Furthermore, when the rats that had been hemisected two months earlier were subjected to a second cut at the same site, chromatolytic neurons were observed in the spinal cord as well as in the motor cortices of both sides. The hemisected rats demonstrated limited recovery in limb movement. The evidence of this study clearly shows that sprouting of nerve fibres has occurred in the lesioned adult rat spinal cord.

Bilateral congenital corneal keloids and anterior segment mesenchymal dysgenesis in a case of Rubinstein-Taybi syndrome.

Purpose. To report the unusual association of bilateral corneal keloids and anterior segment mesenchymal dysgenesis in a child with Rubinstein–Taybi syndrome. Methods. Case report of a 2-year-old boy. Results. Excision of the epicorneal mass in the right eye was followed by recurrence of the lesion. Multiple penetrating keratoplasties were unsuccessful in reconstructing the anterior segment because of recurrent corneal epithelial breakdown, suggesting limbal stem cell insufficiency. Histopathology and electron microscopy of the excised mass lesion showed features typical of a corneal keloid: thickened keratinized epithelium, absent Bowmans layer, and fibrovascular hyperplasia, with haphazard orientation of the collagen lamellae. Ultrasound biomicroscopy and intraoperative findings suggested a diagnosis of Peter anomaly, but genetic analysis did not show a PAX6 mutation. Conclusion. The findings in our patient add to the spectrum of ocular changes described in Rubinstein–Taybi syndrome and confirm earlier reports of poor ocular prognosis in corneal keloids and Rubinstein–Taybi syndrome.

Changes of Cytochemical Markers in the Conjunctival and Corneal Epithelium After Corneal Debridement

Abstract1. The aim of this study was to determine the epithelial changes of the conjunctiva and cornea up to 7 days after corneal debridement and the changes highlighted included (1) proliferation, (2) production of growth factor, (3) changes in calcium binding protein marker, (4) production of cytokine, and (5) maturity of the regeneration corneal epithelium.2. The cytochemical changes of the corneal and conjunctival epithelia of rabbit were analyzed up to 7 days after debridement.3. An increase in proliferating cell nuclear antigen (PCNA) was observed in the limbal epithelia 12 hr after lesion and reached a peak by 48 hr.4. Some proliferating limbal cells also contained epidermal growth factor (EGF) beginning 24 hr after injury. The early limbal cell proliferation and the EGF production and their persistence until 7 days after lesion were likely involved with the process of regeneration.5. Other positive markers appeared after lesion included tumor necrosis factor (TNFα) and calcium binding proteins S100A and S100B, which appeared mainly within the first 48 hr after lesion and then started to decline. The short appearance and the relatively small quantity of TNFα indicated that this cytokine was probably not very important in the repair process and its appearance might be related to the injury induced. The presence of S100A and S100B could be associated with both cell death after injury and the proliferation of new epithelium.6. The cornea epithelium was still immature 7 days after lesion in that it still contained cytokeratin.7. In conclusion, the critical hours of peak conjunctival and corneal changes after corneal debridement were in the first 2 days.

An immunohistochemical study of the c-fos protooncogene in the developing human retina

The localization and distribution of the protooncogene c-fos were studied immunohistochemically in the retina of human fetuses ranging in age from 15 to 40 weeks of gestation. The highest levels of immunoreactivity were observed in the retinae of younger fetuses, decreasing in intensity with increasing age. At 15 weeks of gestation intense immunoreactivity was observed in the inner nuclear layer while the photoreceptor cells exhibited moderate staining. At 26 weeks of gestation, immunoreactivity in the inner nuclear layer was reduced. The ganglion cells, amacrine cells and photoreceptor cells showed moderate immunopositivity throughout the 26-40 weeks period. The role of c-fos in development is discussed in the light of its other known functions.

Metabolic and Phagocytic Activities of the Mouse Retina after 6-Hydroxydopamine Treatment

The activities in mouse retinae were evaluated after 6-hydroxydopamine injection. A significant increase in labeled leucine uptake was evident in the photoreceptor layer. Latex uptake in the pigment epithelium showed no changes. Denervation of catecholaminergic terminals inside the retinae thus affected the metabolism of photoreceptors.

Optic nerve transection and tectal removal affect phagocytic activity of the pigment epithelium in goldfish.

The phagocytic ability of the pigment epithelium after optic nerve sectioning and tectal removal was investigated in the goldfish by the method of ingestion of latex beads. 4 days after sectioning, an increase in latex beads was evident which decreased by the end of the first week. Tectal removal also triggered increase intake of latex beads which was presented after 7 days.

ENKEPHALIN POSITIVE SITES IN THE DEVELOPING HUMAN CEREBELLUM

Enkephalin (ENK) positive sites in the developing human cerebellum (gestation ages 18 weeks to 30 weeks) were studied by immunohistochemistry (ABC method). Positive reactions were registered as early as 20 weeks of gestation and initially in the deep cerebellar nuclei. By 23 weeks some mossy fibers exhibited positivity and by 27 weeks some climbing fibers as well as a few Purkinje basket, golgi and granule cells were also positive. This result indicated that the human cerebellum possesses ENK positive fibers and neurons before birth.

The eyes of anencephalic babies: a morphological and immunohistochemical evaluation.

This study studied the eyes of three anencephalic stillborns to evaluate whether brain degeneration affected eye development and/or survival. The study encompassed histology, scanning electronmicroscopy, and immunocytochemistry. The corneae were otherwise normal except for the presence of blood vessels in the stroma and the posterior surface of the cornea demonstrated wrinkles. Synaechia was present and the lens had occasional vacuolated cells. The retinae had normal layers in most regions except the center where fibroblasts infiltration was observed. The optic nerve was atypical and composed of aggregates of disoriented fibroblasts and disoriented nerve fibers. Anti-cleaved caspase 3 immunocytochemistry revealed only few positive dying cells in the visual cell layer. Antineurofilament 200 reactions demonstrated positive ganglion cells even in the anencephalic eyes. The choroids in anencephaly had more VEGF positive sites, indicating vascularization in both control and anencephalic eyes. If the brains degenerate before retinal maturation, then such degenerations may not have an effect on subsequent retinal development except for the degeneration of the nerve fiber layer. If the brains degenerate after retinal maturation, then the survival of the retinae does not appear to rely on its linkage with the brain at birth, again apart from the degeneration of nerve fibers.