Activation of CPP32-like caspases contributes to neuronal apoptosis and neurological dysfunction after traumatic brain injury.

We examined the temporal profile of apoptosis after fluid percussion-induced traumatic brain injury (TBI) in rats and investigated the potential pathophysiological role of caspase-3-like proteases in this process. DNA fragmentation was observed in samples from injured cortex and hippocampus, but not from contralateral tissue, beginning 4 hr after TBI and continuing for at least 3 d. Double labeling of brain with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and an antibody directed to neuronal nuclear protein identified apoptotic neurons with high frequency in both traumatized rat cortex and hippocampus. Cytosolic extracts from injured cortex and hippocampus, but not from contralateral or control tissue, induced internucleosomal DNA fragmentation in isolated nuclei with temporal profiles consistent with those of DNA fragmentation observed in vivo. Caspase-3 mRNA levels, estimated by semiquantitative RT-PCR, were elevated fivefold in ipsilateral cortex and twofold in hippocampus by 24 hr after TBI. Caspase-1 mRNA content also was increased after trauma, but to a lesser extent in cortex. Increased caspase-3-like, but not caspase-1-like, enzymatic activity was found in cytosolic extracts from injured cortex. Intracerebroventricular administration of z-DEVD-fmk-a specific tetrapeptide inhibitor of caspase-3-before and after injury markedly reduced post-traumatic apoptosis, as demonstrated by DNA electrophoresis and TUNEL staining, and significantly improved neurological recovery. Together, these results implicate caspase-3-like proteases in neuronal apoptosis induced by TBI and suggest that the blockade of such caspases can reduce post-traumatic apoptosis and associated neurological dysfunction.

Activated macrophages in experimental subretinal neovascularization.

Laser-induced subretinal neovascularization (SRN) in monkey retinas was investigated by immunohistochemical techniques to identify the presence and location of activated macrophages. Retinal lesions were examined 3, 8 and 14 days after intensive argon laser treatment, and the distribution of interleukin-1 (IL-1)-containing cells in the lesions was determined by the orthogonal reconstruction of serial sections. Macrophages were present in the subretinal space of day 3 and day 8 lesions. These IL-1-containing cells were distributed about the area of rupture of Bruch's membrane and were quite common in lesions taken 3 days following laser treatment. While still apparent, the number was decreased at 8 days, and none were found 14 days after laser treatment. The temporal and spatial distribution of IL-1-staining macrophages paralleled the development of SRN, suggesting a relationship between the presence of activated macrophages and the initiation of neovascularization in this model.

Fibrovascular proliferation and retinal detachment after intravitreal injection of activated macrophages in the rabbit eye.

Injection of activated macrophages into the posterior vitreous of the rabbit induced vigorous fibrovascular proliferation over the optic disk and medullary rays, as demonstrated by 3H-thymidine autoradiography. One week after injection, endothelial cells and pericytes of the capillaries near the inner surface of the optic disk and rays were labeled; fibroblast-like cells, which were also labeled, migrated and formed vitreous strands. By the second week after injection, the fibrovascular tissue proliferated most actively, and traction medullary ray detachment and peripapillary retinal fold formation were observed. The cellular proliferation was accompanied by inflammatory cell infiltration. Glial cells within the optic disk, as well as retinal pigment epithelial cells beneath the detached retina, were labeled by 3H-thymidine. These results demonstrate that the fibrovascular proliferation originates from the vessel complex of the optic disk and medullary rays in this experimental model of retinal detachment.

Experimental subretinal neovascularization in the rabbit.

Subretinal neovascularization (SRN) in the rabbit was induced by subretinal injection of vitreous without rupture of Bruch's membrane. Eight of 26 eyes developed SRN. The incidence of SRN rose from 33% to 57% in a period of 4-40 weeks. Because of the absence of any fluorescein angiographic indication of SRN, these occult new vessels were identified by light and transmission electron microscopy. Histological examination showed that these newly formed vessels are composed of continuous capillaries with the morphologic characteristics of choriocapillaris, including diaphragmed fenestrations, basement membranes, and junctional complexes. The new vessels originated from the choriocapillaris and penetrated through Bruch's membrane into the subretinal space, where they were associated with the degenerated sensory retina and proliferating glial and/or RPE cells. This experiment provides a model of SRN without breaks in Bruch's membrane.

Morphologic observations of retinal pigment epithelial proliferation and neovascularization in the rabbit.

Neovascularization and proliferation of the retinal pigment epithelium (RPE) was induced in the rabbit by subretinal injection of vitreous without rupture of Bruch's membrane. New vessels developed between the layer of RPE and photoreceptor outer segments, but were enveloped in proliferating RPE. For this reason they were occult; no fluorescein leakage was visible by angiography. The vessels were identified only by histologic examinations. Endothelial cell budding was the initial stage of vessel development, first seen two weeks after injection. The new vessels grew from the choriocapillaris, penetrated Bruch's membrane, and spread into the subretinal space, despite the absence of subretinal fluid. Fenestrations with diaphragms were found in the endothelial walls during the earliest stages of vessel formation, and were also present in the fully matured vessels. Intermediate junctional complexes were frequently observed among the endothelial cells. During maturation of these plexi, junctions changed from open to putative tight junctions. The mature vessels were ultimately completely enveloped by collagen and RPE cells. Our results show that all new vessels in this animal model have the morphologic characteristics of choriocapillaris. We assume that they leak fluorescein, as does the choriocapillaris, but that the dye has no opportunity to pool in the subretinal space and thus cannot be seen during angiography.

Subretinal choroidal neovascularization as a response to penetrating retinal injury in the pigmented rabbit.

Subretinal choroidal neovascularization has been identified in the pigmented rabbit in association with penetrating retinal injury. In this study, the subretinal new vessels were not contained within the fibrous scar, but were located posterior to it and impinged on the visual streak. Histology revealed subretinal neovascularization in the region of transition from normal to atrophic retina. These new vessels originated from the choroidal vessels and extended through a spontaneous break in Bruch's membrane into the retinal pigment epithelium. There was degeneration of the photoreceptors and hyperplasia of the retinal pigment epithelium in the region of neovascularization. The subretinal new vessels identified in the pigmented rabbit have many of the morphological features seen in the human clinical condition, and therefore this animal model may be useful in studying human choroidal neovascularization.

Glial epiretinal membranes and contraction. Immunohistochemical and morphological studies.

It has been suggested that glial cells do not contribute substantially to the contractile forces generated by epiretinal membranes. We have established a rabbit model in which epiretinal membranes form on the inferior peripheral retina after the injection of activated macrophages into the vitreous. By two months, the membranes were extensive but without evidence of traction. At four months, however, full-thickness retinal folds were present beneath the thick epiretinal membrane. A homogeneous glial cell composition was suggested by light microscopic examination of serial sections through several membranes. Immunohistochemical staining with anti-glial fibrillary acidic protein and antivimentin and immunoelectron microscopy confirmed that these thick epiretinal membranes were composed entirely of glial cells, which may cause mild traction on the retina; this traction is associated with cell alignment and the tissue bridges connecting the membrane and the retina. The fusiform densities and indented nuclei suggested that the glial cells within the membrane may possess some characteristics of myofibroblasts.

Breakdown of Bruch's membrane after subretinal injection of vitreous. Role of cellular processes.

It was recently shown that the injection of autologous vitreous beneath the retina of rabbits leads to retinal degeneration, subretinal cellular proliferation and neovascularization. The current study, using electron microscopy, was designed to determine the cellular processes involved in the breakdown of Bruch's membrane in this model. Bruch's membrane was not mechanically damaged by the injection and appeared intact for the first 1 to 2 days after injection. Subsequently, numerous breaks in Bruch's membrane were found associated with invasion of macrophages and fibroblasts; in addition, budding and penetration of retinal pigment epithelial (RPE) and choroidal endothelial cells into Bruch's membrane were noted. Although it was not proven that these cells, per se, were responsible for the breaks, that these cells actively penetrate Bruch's membrane is a reasonable hypothesis.

Experimental changes resembling the pathology of drusen in Bruch's membrane in the rabbit.

Drusen-like changes in Bruch's membrane following subretinal injection of vitreous in the rabbit were studied by electron microscopy. A sequence of changes is seen that closely panellels those observed during drusen formation in primates. The initial event is the budding of retinal pigment epithelial (RPE) cells into Bruch's membrane; the buds, which contain cytoplasm and plasma membrane, are connected to the cytoplasm of the parent RPE cell. Most buds are surrounded by basement membrane, but some penetrate RPE basement membrane into Bruch's membrane. Later, RPE buds completely separate from the parent RPE cell and show degeneration and disintegration. Finally, drusen-like material, including vesicular, granular, tubular and linear structures, is dispersed from the bud remnant into Bruch's membrane. The study described herein thus supports the hypothesis that drusen are the result of budding from the RPE.

Cellular proliferation induced by subretinal injection of vitreous in the rabbit.

A new experimental model of subretinal cellular proliferation, based on injection of autologous vitreous into the subretinal space of rabbits, was studied by light and electron microscopy. As early as five days after injection, proliferation of retinal pigment epithelial (RPE) and retinal glial cells was observed in the subretinal space. These morphologically distinct proliferating cells were sometimes joined by junctional complexes. Morphologically, the proliferating RPE cells resembled either RPE cells or fibroblasts. Some proliferating RPE cells also retained their epithelial characteristics (ie, basement membranes and cell junctions), while others were partially dedifferentiated and showed some embryonic features. New formation of melanin could be identified within the proliferated RPE cells, which could account, in part, for the hyperpigmentation at the site of the bleb caused by the injection of vitreous. The results demonstrated that injection of autologous vitreous into the subretinal space can lead to subretinal proliferation of retinal glial and RPE cells in the rabbit.

Immunofluorescent studies of fibronectin and laminin in the human eye.

The topographic distribution of fibronectin and laminin in young and old human eyes was determined by indirect immunofluorescent techniques. These two glycoproteins may play a role in the attachment of the vitreous to the internal limiting membrane (ILM) and the internal limiting membrane to the Mueller cell processes. A double-laminated pattern of fluorescence for both glycoproteins was frequently found at the ILM of the posterior retina of aged eyes. This pattern of fluorescence, which was rarely seen in young eyes, may represent senescent changes in the ILM which could predispose the eye to posterior vitreous detachment.

Alterations in the distribution of fibronectin and laminin in the diabetic human eye.

The distribution of fibronectin and laminin in diabetic human eyes was determined by indirect immunofluorescent techniques. The intense fluorescence suggests increased amounts of fibronectin and laminin in the diabetic internal limiting membrane (ILM). A double laminated pattern of fluorescence for both glycoproteins suggests structural abnormalities of the ILM of the posterior retina. Preretinal and subretinal proliferative tissues fluoresced strongly and diffusely with antifibronectin. This study indicates that in diabetic patients, the ILM, especially in the posterior retina, is biochemically and morphologically abnormal.

Relative permeability of retina and retinal pigment epithelium to the diffusion of tritiated water from vitreous to choroid.

The movement of intravitreally injected tritiated water from the vitreous to the choroid was accelerated by the removal of intervening retina. Both rate of transfer and peak choroidal levels of the tracer were increased, but the proportion of the intravitreal dose recovered was unaltered. In contrast, the movement of tritiated water after diffuse damage to the retinal pigment epithelium by sodium iodate was similar to that of control eyes. The main resistance to the diffusion of this tracer from the vitreous to the choroid is the retina. The differential permeability of the retina and the retinal pigment epithelium may have a role in normal retinal adhesion.