Biomedical subjects

M del Cerro

Publications and source records attributed to M del Cerro.

At least 37 records · Page 2Linked to original sources

PubMed1995

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.

Aging↗

PubMed1995

The intensity of the pupillary light reflex does not correlate with the number of retinal photoreceptor cells.

The purpose of this study was to determine if the pupillary light reflex (PLR) can serve as an indicator of the number of photoreceptor cells present in the rat retina to a sufficient degree of precision to be useful for testing the functional effects of retinal transplantation. The PLR was measured as percentage constriction of normal Fischer 344 rats (n = 14) and compared to the PLR of light-damaged (1300 luxes/30 days exposure) Fischer rats (n = 13). Additionally, the PLR of RCS-rdy+ (congenic) rats (n = 8) was compared to the PLR or RCS dystrophic rats (n = 7). Three eyes from each group were randomly chosen for morphometry. The number of photoreceptor nuclear profiles per 60 microns of retinal length was counted at six predetermined loci and averaged. The mean PLR of Light-Damaged F344 group (64%) was significantly different from the mean PLR of the Normal F344 group (75%) (P = 0.003). However, the mean PLR of the RCS Dystrophic group (72%) did not differ from the mean PLR of the RCS Congenic group (71%) (P = 0.82). Morphometry revealed that the mean number of photoreceptor nuclear profiles within each group of animals was vastly different: Normal F344 = 138, Light Damaged F344 = 19, Congenic RCS = 93, and Dystrophic RCS = 1. No correlation was found between intensity of PLR and number of photoreceptors present (r = 0.11, P = 0.78).(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1995

Evaluation of tissue adhesives in closure of scleral tunnel incisions.

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 self-sealing 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.

Analysis of Variance↗

PubMed1995

Effects of the picosecond neodymium:YLF laser on poly(methyl methacrylate) intraocular lenses during experimental posterior capsulotomy.

We evaluated the effect of the neodymium:YLF picosecond laser on intraocular lenses (IOLs) during experimental posterior capsulotomy and established the minimum damage threshold for lathe-cut poly(methyl methacrylate) (PMMA) IOLs. Single-piece, biconvex, lathe-cut PMMA lenses were placed in a model eye chamber against a latex membrane with a central area of lens-capsule touch. Energy levels from 40 micro J to 360 microJ pulse (0.4 mJ to 3.6 mJ) with a 0.5 mm line and a spot pattern were tested using different focal offsets. Threshold damage level was determined under the scanning electron microscope after observing the minimal energy level that did not cause damage to or pits in the IOLs. Lenses were then analyzed for size and type of damage. The 0.5 mm line pattern at 160 microJ using 350 microns offset was safe and effective for posterior capsulotomy in vitro. spot pattern caused IOL damage in the form of small pits at much lower energy levels of 80 microJ using 400 microns offset than the line pattern.

Lasers↗

PubMed1995

An improved method of isolating fetal human retinal pigment epithelium.

The purpose of this study was to develop an improved method of isolating fetal human retinal pigment epithelium (RPE) for tissue culture or transplantation. Fetal human eyes ranging from 8 to 20 wks of gestation were collected and stored in Optisol solution. Under a dissecting microscope, an incision was made behind the ora serrata and extended circumferentially to remove the anterior segment. The vitreous was withdrawn, and the neural retina was carefully detached from the RPE. The sclera then was teased away from the choroid-RPE. The choroid-RPE was treated with 2% dispase in DMEM + 20 mM HEPES at 37 degrees C for 25 min. While still in dispase, the RPE was separated from the choroid using a pair of fine tipped jeweler's forceps under dark-field. An intact sheet of RPE could be separated from the choroid after treatment with dispase. No choroidal contamination was present as determined by light microscopy or cell culture. In vitro, the isolated RPE cells demonstrated classic cobblestone phenotype and expressed cytokeratin. This technique provides an easy and reliable method for isolating pure sheets of fetal human RPE. It also allows utilization of the neural retina of the same eye for other purposes, as the neural retina is not exposed to the enzymatic digestion. These features make this method especially useful for RPE and retinal transplantation; such an application is already underway.

Cell Separation↗

PubMed1995

A functional analysis of the age-related degeneration in the Fischer 344 rat.

We have previously described how the expression of photoreceptor cell degeneration in the Fischer 344 rat is affected by age, retinal topography, and gender. Degeneration in the central and equatorial regions progresses linearly with age throughout the life span of the animal, while the periphery of the male is subject to sudden and dramatic losses of cells in the superior hemisphere after 12 months and in the inferior hemisphere after 18 months of age. The purpose of the present study was to determine how this degeneration affected retinal function and visual ability in the male Fischer 344 from 3 to 24 months of age. Functional testing included the electroretinogram (ERG, measuring both a-wave and b-wave amplitudes and implicit times) and the behavioral method of startle reflex modification (RM, which measures the degree to which a light flash inhibits the response to an immediately subsequent loud noise). All of the functional measures showed a decline with age, but varied in their time course. ERG amplitudes showed a linear decline in amplitude over the entire age range. In contrast, the implicit times of the ERG waves and the degree to which the light flash inhibited the startle reaction both showed a slight maturation in function (faster implicit times and greater inhibition) from 8 or 12 months of age. After 18 months of age, the implicit time showed a significant increase and the startle response showed a significant decrease. This study shows how visual function correlates with the histopathological changes seen in age-related retinal degeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1995

[Correlation between corneal sensitivity and nerve regeneration following excimer laser ablation].

To compare the recovery of corneal sensitivity and corneal regeneration following excimer laser and manual epithelial debridement, the corneal epithelium of the right eye of New Zealand white rabbits (n = 21) was manually debrided and the left eye was ablated with the excimer laser (47 microns depth, 5 Hz, and 160 mJ/cm2). We observed the wound healing rate and corneal sensitivity. The relative density of innervation on the intraepithelial layer was measured using gold chloride staining or right microscopic examination for 30 weeks. In the laser-ablated cornea sensitivity rapidly increased to a normal level by day 5, and then it continued to increase gradually and reached a maximum at day 42. Thereafter it remained elevated to 126 days and returned to normal at 210 days. There were significant differences in the recovery of sensitivity following excimer ablation and manual epithelial removal. The relative density of innervation on the intraepithelial layer after excimer ablation was significantly higher than manual debridement at day 35. We observed a correlation between increased sensitivity levels and increased nerve density. This result suggests that for photorefractive keratectomy or phototherapeutic keratectomy, manual debridement of the corneal epithelium should be performed before stromal excimer ablation.

Animals↗

PubMed1994

The influences of age, retinal topography, and gender on retinal degeneration in the Fischer 344 rat.

The Fischer 344 (F344) rat is presently the animal of choice for age-related research. The existence of an age-related retinal degeneration was reported previously in the males of this strain, but a gender comparison has not been performed. In this study, histological and morphometric measurements of the retina related to age, retinal topography, and gender were made on 3- to 24-month-old animals. The thicknesses of the outer nuclear layer (ONL) and the photoreceptor layer (PRL) were measured from sagittal sections at six loci. Retinas of both sexes showed steady decline with age in the thicknesses of the ONL and PRL at all locations. An important finding was the presence, after 12 months of age, of a drastically accelerated rate of peripheral retinal degeneration seen only in male subjects. Females showed a less dramatic rate of peripheral degeneration which did not begin until after 18 months of age. In addition, two other forms of retinal degeneration were found--cystoid degeneration was found earlier and more frequently in the male, while a paving-stone type of degeneration was found in both sexes. These two types of lesions were preferentially, but not exclusively found in the peripheral retina. In conclusion, the F344 rat offers a convenient model to study a pattern of retinal degeneration affected by the combination of gender, regional and age-related factors.

Aging↗

PubMed1994

Retinal ganglion cell survival is promoted by genetically modified astrocytes designed to secrete brain-derived neurotrophic factor (BDNF).

Genetically engineered cells carrying genes for neurotrophic factors have potential application for treatment of neurodegenerative diseases and injuries to the nervous system. Brain-derived neurotrophic factor (BDNF) promotes the survival of specific neurons, including retinal ganglion cells (RGC). To determine whether genetically engineered astrocytes might be used for delivering bioactive BDNF, we infected primary type 1 rat astrocytes with a retrovirus harboring a human prepro-BDNF cDNA and assayed the medium conditioned by these astrocytes for effects on survival of rat RGCs in vitro. High levels of BDNF mRNA were expressed by infected astrocytes, but not by control astrocytes as determined by RNase protection assay using a BDNF specific probe. To test for secretion of bioactive BDNF from the transgenic astrocytes, embryonic day 17 rat retinas were dissociated and grown in medium conditioned (CM) for 24 h by astrocytes infected with a replication deficient retrovirus carrying BDNF, NGF, or alkaline phosphatase (AP) cDNA. After 3 days, the number of Thy-1 immunoreactive RGCs was counted. BDNF astrocyte CM significantly enhanced RGC survival by 15-fold compared to the AP control. NGF astrocyte CM had no significant effect. The rate of BDNF secretion was estimated at 83-166 pg/10(5) cells/h. This study demonstrates that astrocytes can be genetically engineered to synthesize and secrete bioactive BDNF. These techniques may be applicable to rescuing neurons from degenerative processes and also for enhancing their survival following transplantation.

Animals↗

PubMed1994

A test of the hypothesis that diagnostic ultrasound disrupts myelination in neonatal rats.

Neonatal rats were exposed or sham exposed for 30 min to pulsed ultrasound [2.25 MHz carrier frequency, 1 microsecond pulse length, 50 Hz pulse repetition frequency (PRF), 50 W/cm2 Imax, 2 mW/cm2 ITA], euthanised and prepared for electron microscopic analysis of the nodes of Ranvier of the dorsal and ventral roots of the spinal cord. There was also a cage control. All materials were processed and scored blindly, evaluating whether perinodal myelin was normal. Rats from all regimens had areas of disrupted myelination. There was no statistically significant difference among the regimens for absence of myelination. The results did not confirm an earlier report that diagnostic ultrasound disrupts myelination in neonatal rats.

Animals↗

PubMed1994

Corneal sensitivity and nerve regeneration after excimer laser ablation.

We studied the recovery of corneal sensitivity and corneal regeneration after excimer laser and manual epithelial debridement. The corneal epithelia of right eyes of New Zealand white rabbits (n = 21) were manually debrided and the corneal epithelia of the left eyes were ablated with the excimer laser (47 mm depth, 5 Hz, and 160 mJ/cm2). The relative density of innervation on the intraepithelial layer was measured using gold chloride staining and light microscopy. Wound healing and corneal sensitivity also were observed. Laser-ablated corneal sensitivity increased rapidly to a normal level by day 5, then increased gradually and reached a maximum at day 42. It remained elevated until 126 days, then returned to normal at 210 days. There were significant differences in the recovery of sensitivity after excimer ablation and manual epithelial removal. At day 35, relative density of innervation in the intraepithelial layer treated with excimer laser ablation was significantly higher than that treated with manual debridement. We observed a correlation between increased sensitivity and increased nerve density after excimer ablation. Compared with manual debridement, laser treatment resulted in an increase in networks of axons with terminal endings. This may not be the only factor correlating directly with hypersensitivity after corneal debridement, but it may indicate a quicker recovery route by using a presurgical manual debridement technique.

Animals↗

PubMed1994

A new procedure for fundus photography and fluorescein angiography in small laboratory animal eyes.

Increasing interest in retinal research demands continuous improvement of experimental techniques and interpretation. Thus, the purpose of our research was to devise a new method for funduscopic photography and fluorescein angiography in the normal or diseased retina of the small laboratory animal that would produce results comparable in optical quality and field coverage to those obtained in human clinical practice. To enhance the view of the small eye, a 2.2 Volk Panretinal lens was held in apposition to the lens of a clinical fundus camera, the Topcon TRC 50FT, by means of a custom made metal sleeve. Albino mice, albino rats, and pigmented rats were photographed. Fluorescein angiography was performed on pigmented rats. Fluorescein was administered intravenously via the jugular vein at a dose of 5 mg/kg. Various speeds of film and flash settings were used depending on the light source and the pigmentation of the animal. Attachment of the 2.2 Panretinal lens to the clinical fundus camera allowed for more clearly defined fundus photographs of the small laboratory animal, as well as an enlarged field of observation over conventional techniques. Consequently, angiography fields and stages documented in the small laboratory animal approximated those obtained in human clinical practice. This technique facilitates the visualization of small fundi and it allows for a fuller documentation of experimental retinal models.

Animals↗

PubMed1994

Differentiation of Y79 retinoblastoma cells with pigment epithelial-derived factor and interphotoreceptor matrix wash: effects on tumorigenicity.

We investigated the in vivo differentiation potential of Y79 human retinoblastoma cells following pre-treatment with two novel neurotrophic agents: PEDF (human recombinant pigmented-epithelial derived factor) or IPM (interphotoreceptor matrix) wash. These agents were able to induce a significant degree of morphological differentiation in vitro. However, 48 days after subretinal transplantation of pre-treated cells, massive tumor formation was apparent. In contrast, Y79 cells pre-treated with retinoic acid/sodium butyrate, which attain a lesser degree of morphological differentiation, did not produce tumors over a 30 to 60 day-survival time (del Cerro et al., Brain Research, 12-22, 1992). We conclude that for PEDF and IPM, the degree of in vitro differentiation and the degree of mitotic arrest are independent features.

Animals↗

PubMed1994

Cytomegalovirus infection of human retinal tissue: an in vivo model.

BACKGROUND: Cytomegalovirus (CMV) retinitis is a common, devastating complication of AIDS. Strict host specificity of human CMV (HCMV), has limited the study of the virus. The purpose of this research was to create a model that allowed the in vivo infection of human retinal tissue with HCMV. EXPERIMENTAL DESIGN: Small fragments of 13-week human fetal retina were transplanted into the anterior chambers of immune-deficient mice. One week later, 15 of 30 grafts were inoculated with HCMV. Infected and noninfected specimens were compared for histopathologic changes at 14, 30, and 60 days at light and electron microscopic levels. Histochemistry was performed to characterize: level of graft differentiation, range of viral replication cycle, and effects on neuronal, glial, and monocytic elements of the retina. RESULTS: At 30 and 60 days, grafts were well differentiated histologically and histochemically. The infected grafts showed numerous large cells containing intranuclear and intracytoplasmic inclusions. Ultrastructural examination revealed viral particles within cytoplasm and nuclei. Infected cells expressed proteins representative of all phases of the HCMV replication cycle. Most HCMV-infected cells expressed the neuronal marker, protein gene product 9.5. In a second series of experiments, tissue from one graft harvested at 45 days postinfection was used to recover HCMV in fibroblast cultures and also used successfully to passage the virus to two control grafts. CONCLUSIONS: This model allows HCMV to actively infect and replicate within highly differentiated human neural tissue. It produces pathology characteristic of CMV-infected neural tissue in a time course suitable for long-term study. The nature of this model will allow the study of: the replication properties of HCMV in nervous tissue, the specific effects of HCMV on the human retina, the interaction of HCMV with other viruses, e.g., HIV, and various anti-viral therapies.

Animals↗

PubMed1993

Surface quality of excimer laser corneal ablation with different frequencies.

To test the effects of different frequencies of a 193-nm excimer laser on the surface smoothness of the ablated materials and the damage to the adjacent structure, four different frequencies (5, 10, 15, and 20 Hz) of a 193-nm excimer laser were used to perform ablations on 20 rabbit corneas and four polymethylmethacrylate blocks at a fluence of 160 mJ/cm2. Each frequency was tested five times on five corneas. The ablated materials were processed and examined with scanning electron microscopy (SEM) and light and transmission electron microscopy (TEM) and a Zygo interference microscope, which quantitatively evaluates the surface smoothness. The results from the Zygo microscope show that there is no statistically significant difference in surface smoothness between any two different frequencies. The SEM reveals similar regularity and uniformity on the ablated surfaces, with no relationship between the laser frequencies and the amount of surface deposits. The TEM demonstrates no correlation between the various frequencies and the thickness of the superficial pseudomembrane and the amount of collateral damage in the adjacent stroma. It appears that the higher frequencies are comparable to the lower one (5 Hz) as to ablation quality, with the benefit of curtailing surgical time and decreasing the chances of eye movement.

Animals↗

PubMed1993

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.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1993

Transplantation of Y79 cells into rat eyes: an in vivo model of human retinoblastomas.

PURPOSE: To develop an in vivo model of human retinoblastoma by returning cultured Y79 retinoblastoma cells to the retinal environment of a widely available laboratory animal. In so doing, to study the survival, integration, and invasive characteristics expressed by tumoral cells grafted into an intraocular milieu from which these progenitor cells originated more than 20 years ago. METHODS: Using the retinal grafting method of Lazar and del Cerro, Y79 cells were injected under direct visualization into the subretinal space of Fischer 344 rats. The host rats included 36 animals that received daily injections of cyclosporin A and 4 that did not. All hosts were sacrificed 30 to 60 days after transplantation. RESULTS: Clinical examination showed vitreal invasion by masses of flocculent white material or the intravitreal formation of solid tumors. Histologic examination showed these formations to be outgrowths of grafted tumoral cells into the host retina and vitreal cavity. Highly anaplastic tumoral cells were also found lodged in subretinal and intraretinal locations. There were signs of continued and intense cell division within the grafts, with no indication of cell-mediated host reaction against the grafted cells. CONCLUSIONS: After intraretinal xenografting, human Y79 retinoblastoma cells retain a highly tumoral nature despite many years of in vitro propagation. When xenografted, these cells survive, grow, and express their malignancy within the retina of the common laboratory rat protected by a moderate immunosuppressive regimen. This partial immunosuppression is a requirement for the xenografts to prosper. This model offers a valuable opportunity to study in vivo the cellular and molecular biology of this and other human retinoblastomas, and it may facilitate the evaluation of antitumoral treatments.

Animals↗

PubMed1992

Intraretinal xenografts of differentiated human retinoblastoma cells integrate with the host retina.

We report on the successful use of chemically modified Y79 human retinoblastoma cells for intraretinal xenografting into damaged adult mammalian eyes. Y79 cells were exposed in vitro to retinoic acid/butyrate to induce differentiation. Using a multisite transplantation method, the suspension was injected into the subretinal space of Fischer 344 rats. The survival, integration, and differentiation potential of these cells was studied, following their return to the intraocular milieu from which the progenitor cells originated. The grafted cells survived and differentiated into immature photoreceptor elements in the subretinal and intraretinal locations, as multiple clusters of rosette-forming cells intimately attached to the host neuroretina. The differentiation process included development of synaptic connectivity of the ribbon type with the surrounding neuropil. No signs of renewed cell division were found within grafts performed on 42 rat eyes, and there was no indication of cell-mediated host reaction against the transplants. This study indicates that tumorigenicity can be suppressed in mitotically arrested Y79 cells, and that these cells are capable of undergoing differentiation in vivo. This provides evidence of the remarkable differentiation properties of human retinoblastomas while indicating that Y79 cells may ultimately be able to substitute for fetal cells in experimental retinal transplantation.

Animals↗