Manuel del Cerro

Neurotoxic Effects of Tumor Necrosis Factor Alpha in Primary Human Neuronal Cultures are Mediated by Activation of the Glutamate AMPA Receptor Subtype: Implications for AIDS Neuropathogenesis

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system is characterized by neuronal loss in discrete areas of the central nervous system. We have previously demonstrated that HIV-infected monocytes in culture with astroglial cells produce high levels (> or = 200 pg/ml) of the cytokine tumor necrosis factor-alpha (TNF alpha). We now demonstrate that TNF alpha (> or = 200 pg/ml) is neurotoxic to cultured primary human fetal cortical neurons at both light and electron microscopic levels. Subtoxic doses of TNF alpha (50 pg/ml) are neurotoxic in combination with the glutamate (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) subtype receptor agonist AMPA (100 microM). The neurotoxic effects of TNF alpha (200 pg/ml) are blocked in part by the AMPA receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (10 microM). This suggests that TNF alpha may exert neurotoxic effects on human neurons by indirect activation of AMPA receptors, which may be important in the pathogenesis and treatment of HIV-mediated encephalopathy.

Allogenic fetal retinal pigment epithelial cell transplant in a patient with geographic atrophy.

PURPOSE To test the hypothesis that healthy fetal retinal pigment epithelium (RPE) can rescue the remaining viable RPE and choriocapillaries and thereby the photoreceptors in non-neovascular age-related macular degeneration (ARMD) (geographic atrophy [GA]). METHODS A 65-year-old legally blind woman with non-neovascular ARMD underwent fetal RPE transplantation. Best-corrected visual acuity testing, detailed fundus examination, fundus photography, fluorescein angiography, scanning laser ophthalmoscope macular perimetry, and humoral and cellular immune response testing were performed. A suspension of RPE was infused into the subretinal space through a retinotomy along the superotemporal arcade at the edge of the area of GA. The patient did not take systemic immunosuppressants. RESULTS The patients vision remained unchanged for 5 months after the surgery. Fluorescein angiography after transplantation showed leakage and staining at the level of the outer retina. There was progressive subretinal fibrosis in the area of the transplant. Immune response studies showed a weakly positive mixed lymphocyte response against phosducin and rhodopsin. CONCLUSION Although it is surgically feasible to transplant fetal RPE to the subretinal space of patients with GA, such an allogenic RPE transplant without immunosuppression leads to leakage on fluorescein angiography and eventual fibrosis. A very weak immune response against proteins associated with photoreceptors is also of concern.

The Transplantation of Human Fetal Neuroretinal Cells in Advanced Retinitis Pigmentosa Patients: Results of a Long-Term Safety Study ☆

The purpose of this study was to determine the long-term safety of transplanting human fetal neuroretinal cells (14 to 18 week gestational age) into a series of patients with advanced retinitis pigmentosa (RP). After obtaining informed consent, both hosts and mothers of donors were screened for transmissible diseases. Pre- and postoperative clinical exams, visual acuity, electroretinograms, and fluorescein angiograms were performed and visual field testing was attempted in each case. Surgically, an anterior approach through pars plana ciliaris was used. A retinotomy was performed in the paramacular area and a two-function cannula was introduced into the subretinal space to deliver a suspension of donor cells. The cell suspension carried approximately 4000 cells/microl; the volume injected did not exceed 150 microl. The patients were examined for periods ranging from 12 to 40 months posttransplantation. To date, no evidence of inflammation, infection, or overt rejection of the graft was noted in the host eye, neither was any change observed in the contralateral, unoperated eye. In conclusion, neuroretinal cells were injected into the subretinal space of 14 patients with advanced RP with no clinical appearance of detrimental effects at the time of surgery or up to 40 months postinjection except in 1 patient who developed retinal detachment. This sets the stage for a phase II clinical trial to determine the possible beneficial effects of this procedure in patients blinded by degenerative retinal disease.

Prenatal development of Bergmann glial fibres in rodent cerebellum.

SummaryThe external granular and molecular layers in the foetal cerebellar cortex of mice and rats were examined by electron microscopy for the presence of Bergmann glial fibres. Morphologically distinct Bergmann fibres were observed at embryonic day E 15 in the mouse and at E 17 in the rat. Even at prenatal stages of development these fibres have a considerable degree of cytological differentiation which permits their identification as glial elements. The glial fibres contain numerous microfilaments, some smooth endoplasmic reticulum, a few mitochondria and scant free ribosomes. They penetrate the molecular and external granular layers radially and terminate with endfeet at the cerebellar surface. The proliferative cells of the external granular layer possess cytoplasmic processes which are oriented randomly, do not have endfeet, and are morphologically distinct from the Bergmann fibres with which they intermingle. In conclusion, immature Bergmann glial cells are present well before birth in the rodent cerebellum.

A new procedure for multiple intraretinal transplantation into mammalian eyes

A new method of intraretinal grafting is described which avoids opening the globe and allows direct visual control of the transplant by observing placement of the graft through the host pupil. A microsyringe with a bevelled needle partially ensheathed in plastic tubing in order to limit penetration was used to inject dissociated donor cells into 2 to 4 pre-selected points of the sub-retinal space of adult rats. The needle tip was inserted through the scleral and choroidal tissue, and observed through the animals pupil as it reached into the sub-retinal space. Donor cells were then injected into several sites of the retina. Results, using ophthalmoscopy as well as light and electron microscopy, confirmed the atraumatic nature of this technique and the survival of grafted cells. Intraretinal injection of colloidal carbon visually illustrated the effectiveness of this method in covering an extended portion of the host retina. The procedure, which is virtually free of complications has enabled us to perform multisite intra-ocular grafting in a safe, easy, and reliable fashion, under direct visual control.

Effect of 2450 MHz microwave energy on the blood-brain barrier to hydrophilic molecules. D. Brain temperature and blood-brain barrier permeability to hydrophilic tracers

Measurement of temperature within the cerebral cortex, hypothalamus, cerebellum and medulla of rats sham-, heat- or microwave-exposed revealed the presence of a thermal gradient within the brain. In all groups, cerebral cortex and the cerebellum were cooler than the deeper hypothalamus and medulla. Exposure to 2450 MHz CW microwaves or ambient heat (42 +/- 2 degrees C) resulted in measurable elevation of regional brain temperature, but without alteration of temperature gradients normally observed within the brain. Exposure to 20 mW/cm2 (SAR approximately equal to 4 W/kg) for 30, 90 or 180 min induced a small, but significantly (U = 0, P less than 0.05) increased temperature of the colon, and in each region of the brain studied. Exposure to an incident power density of 65 mW/cm2 (SAR approximately equal to 13.0 W/kg) for 30 or 90 min or to ambient heat (42 +/- 2 degrees C) for 90 min resulted in a substantially greater thermal response as indicated by higher colonic and brain temperatures. Comparison of regional brain temperature with individual colonic temperatures is expressed as delta T = t degrees Cbrain--t degrees Ccolon. In general delta T values for ambient heat or microwave-exposed rats did not differ significantly from those of sham-exposed animals. Exposure to microwaves or ambient heat did not alter the general relationships between regional brain and colonic temperatures, i.e., cortical and cerebellar temperatures were always below and hypothalamic and medullary temperatures always above corresponding colonic temperatures. The plotted temperature data (brain vs colonic temperature) indicate a linear relationship between brain and colonic temperatures. Levels of sodium fluorescein (NAFl), horseradish peroxidase (HRP) and [14C]sucrose (described in preceding papers) within the brain show a high correlation (P less than 0.05) with brain temperature. Suppression of blood-brain barrier permeability to hydrophilic tracers was most pronounced at brain temperatures exceeding approximately 40 degrees C and is demonstrated to be temperature dependent.

Müller cell changes precede photoreceptor cell degeneration in the age-related retinal degeneration of the Fischer 344 rat

Previously, we have used descriptive pathology and histomorphometry, as well as functional testing to characterize the age-related retinal degeneration in the Fischer 344 rat. These studies suggested an association between Müller cells and photoreceptor cells in this process. The purpose of the present study was to further investigate the respective roles of these cell types in the development and progression of the retinal degeneration. Retinas from male Fischer 344 rats aged 3-24 months were first studied by light and electron microscopy. Since Müller cells abundantly express GFAP during pathological states, GFAP content was studied by immunocytochemistry and by immunoblotting following one- and two-dimensional gel electrophoresis. Microscopically, at 12 months, Müller cells showed a gradient of immunoreactivity for GFAP that was minimal in the central retina, positive for their radial processes in the equator, and abundantly expressed in the periphery. At this age, the photoreceptor cells were just beginning to degenerate in the far periphery, while they appeared healthy in the equatorial and central regions. By 24 months, Müller cell hypertrophy was seen in the peripheral regions where photoreceptor cell degeneration was most severe, while the immunoreactivity of the Müller cell processes spread further toward the central regions, ahead of the degeneration of the photoreceptor cells. Thus, Müller cell changes actually preceded photoreceptor degeneration in time and location. This phenomenon was confirmed by measurement of GFAP after one- and two-dimensional PAGE. These findings show that Müller cell changes precede chronic photoreceptor cell degeneration in the aging Fischer 344 rat and are consistent with the hypothesis that Müller cell alteration may be the primary mechanism of this age-related retinal degeneration.

Peptidergic and catecholaminergic fibers in the human corneal epithelium. An immunohistochemical and electron microscopic study.

Abstract Innervation of the clinically normal human corneal epithelium was investigated utilizing immunohistochemical and electron microscopic techniques. All corneal epithelial sheets examined demonstrated neuron specific enolase (NSE: a non‐specific marker for neural elements), calcitonin gene‐related peptide (CGRP: a putative marker for sensory fibers), and tyrosine hydroxylase (TH: a marker for catecholaminergic nerves) immu‐noreactive fibers. NSE, CGRP, and TH fibers formed a dense basal epithelial plexus. The CGrp fibers tended to have beaded profiles, while TH fibers were smooth. Numerous free nerve endings originating from the basal epithelial plexus og NSE and CGRP fibers terminated throughout the thickness of epithelium. The densities of fibers in the basal epithelial nerve plexus were: NSE > CGRP > TH. Transmission electron microscopy demonstrated two types of epithelial nerve fibers, one containing large dense‐core vesicles and another small dense‐core visicles. Both types contained clear vesicles. These large and small dense‐core vesicle fibers appeared to correspond to the CGRP and TH immunoreactive fibers, respectively. These results provide morphological baseline data on the normal sensory and sympathetic corneal epithelial innervation.

Intraretinal transplantation of fluorescently labeled retinal cell suspensions

Dissociated cell suspensions of neonatal neural retina, labeled with the fluorescent dyes Fast blue or Fluoro-gold, were transplanted into the retina of normal adult rats or of rats affected by late stage phototoxic retinopathy. Light microscopy showed good survival, differentiation, and integration of the transplants, as well as permanence of the label up to 100 days. The results indicate that the transplantation of dissociated, fluorescently labeled retinal cells has a number of advantages over the transplantation of solid fragments of retinal tissue, previously performed by ourselves and others. The following are some of the most immediate procedural advantages: the number of transplanted cells can be assessed, the transplanted cells are in a more intimate contact with the host tissue and therefore integrate better with the host, and the fluorescent tags permit precise determination of the survival and distribution of the transplanted cells.

Evaluation of tissue adhesives in closure of scleral tunnel incisions

Abstract Using a biomechanical wound strength model, we compared the efficacy of cyanoacrylate and fibrin glues used to close scleral tunnel incisions. Scleral tunnel incisions were made in four groups of rabbits: (1) traditional selfsealing incision, (2) modified non‐self‐sealing incision, (3) method 2, closed with cyanoacrylate glue, or (4) method 2, closed with fibrin glue. Overall, Groups 1 and 4 showed the least clinical reaction, the slightest decrease in intraocular pressure (which recovered to baseline by day 7), and the most significant recovery of postoperative astigmatism. Initially, the bursting pressure in Groups 1 and 3 was statistically the highest (P < .005). By day 3, wound strengths in Groups 1 and 4 were comparable. Bursting pressure decreased in Groups 2 and 3 by day 7. Our results indicate that clinical responses, intraocular pressure, induced astigmatism, and ultimately wound strength were comparable in fibrin‐glue‐closed scleral pocket and sutureless self‐sealing cataract incisions. Although cyanoacrylate glue cures immediately and initially demonstrates a strong adhesive quality, it causes a severe inflammatory response that inhibits subsequent collagen remodeling. Fibrin tissue adhesives may have an application as adjunctive means of closing scleral tunnel incisions.