Optimization of immunocytochemistry in cytology: comparison of two protocols for fixation and preservation on cytospin and smear preparations.

OBJECTIVE: A new protocol for fixation and slide preservation was evaluated in order to improve the quality of immunocytochemical reactions on cytology slides. METHODS: The quality of immunoreactions was evaluated retrospectively on 186 cytology slides (130 direct smears, 56 cytospins) prepared from different cytology samples. Ninety-three of the slides were air dried, stored at -20 °C and fixed in acetone for 10 minutes (Protocol 1), whereas the other 93 were immediately fixed in methanol at -20 °C for at least 30 minutes, subsequently protected with polyethylene glycol (PEG) and stored at room temperature (Protocol 2). Immunocytochemical staining, with eight primary antibodies, was performed on a Ventana BenchMark Ultra instrument using an UltraView Universal DAB Detection Kit. The following parameters were evaluated for each immunoreaction: morphology preservation, intensity of specific staining, background and counterstain. The slides were blinded and independently scored by four observers with marks from 0 to 20. RESULTS: The quality of immunoreactions was better on methanol-fixed slides protected with PEG than on air-dried slides stored in the freezer: X = 14.44 ± 3.58 versus X = 11.02 ± 3.86, respectively (P < 0.001). CONCLUSION: Immediate fixation of cytology slides in cold methanol with subsequent application of PEG is an easy and straightforward procedure that improves the quality of immunocytochemical reactions and allows the storage of the slides at room temperature.

Evaluation of the effectiveness of mass trapping with BG-sentinel traps for dengue vector control: a cluster randomized controlled trial in Manaus, Brazil.

The objective of this study was to assess the effectiveness of BG-Sentinel (BGS) traps for mass trapping at the household level to control the dengue vector, Aedes aegypti (L.), in Manaus (Brazil) by performing a cluster randomized controlled trial. After an initial questionnaire and baseline monitoring, 6 out of 12 clusters were randomly allocated to the intervention arm, where participating premises received one BGS trap for mass trapping. The other six clusters did not receive traps and were considered as the control arm. Biweekly monitoring with BGS in both arms assessed the impact of mass trapping. At the end of the study, a serological survey was conducted and a second questionnaire was conducted in the intervention arm. Entomological monitoring indicated that mass trapping with BGS traps significantly reduced the abundance of adult female Ae. aegypti during the first five rainy months. In the subsequent dry season when the mosquito population was lower, no effect of mass trapping was observed. Fewer Ae. aegypti females were measured in the intervention arm during the next rainy period, but no significant difference between arms was observed. The serological survey revealed that in participating houses of mass trapping areas recent dengue infections were less common than in control areas, although this effect was not statistically significant. The majority of participants responded positively to questions concerning user satisfaction. Our results suggest that BGS traps are a promising tool which might be deployed as part of dengue control programs; however, further investigations and larger scale studies are necessary.

Diagnostic yield of muscle biopsies in pediatric population: a tertiary center experience. / Rendimiento diagnóstico de las biopsias musculares en la población pediátrica: experiencia de un centro terciario.

BACKGROUND AND AIM: Muscle biopsy is still an important exam on the investigation of neuromuscular diseases although data regarding its diagnostic yield can be disappointing. We aimed to analyze the diagnostic yield of muscle biopsies in the pediatric population. PATIENTS AND METHODS: We retrospectively analyzed a tertiary Neuropathology laboratory database to identify patients (<18 years old), submitted to muscle biopsy between January 2015 and August 2019. Demographics, clinical presentation, diagnosis, treatment, and follow-up were evaluated. Descriptive statistical analysis was performed. RESULTS: One-hundred and six patients were included, 52,8% (n = 56) were male. Median age at biopsy was 6 years (IQR 10 years). Patients were divided into 8 groups, according to clinical diagnostic suspicion: mitochondrial myopathies (n = 29), congenital myopathies (n = 9), inflammatory myopathies (n = 8), muscular dystrophies (n = 7), raised CK values in serum (n = 7), metabolic myopathies (n = 5), weakness /other neuromuscular symptoms (n = 30) and multiple clinical suspicions (n = 11). Biopsy was normal in 50 patients. Of the remaining, 27 displayed specific diagnostic features, with 88,9% (n = 24) allowing a definite diagnosis: muscular dystrophies (n = 7), metabolic myopathies (n = 5), congenital myopathies (n = 4), inflammatory myopathies (n = 4), mitochondrial myopathies (n = 3) and spinal muscular atrophy (n = 1). Histology led to a change of treatment in 4 patients, all diagnosed with inflammatory myopathies. Median length of follow-up was 1 year (IQR 2 years). CONCLUSION: Biopsy diagnostic yield was 22,6%, and it was useful either in diagnostic or therapeutic approaches in 35,8%. Although advances of molecular techniques led to a decrease in muscle biopsy indications, it remains an important tool on the diagnosis of neuromuscular diseases.

TITLE: Rendimiento diagnóstico de las biopsias musculares en la población pediátrica: experiencia de un centro terciario.Introducción. La biopsia muscular es un examen importante en la investigación de enfermedades neuromusculares, aunque su rendimiento diagnóstico puede ser decepcionante. Objetivo. Analizar el rendimiento diagnóstico de las biopsias musculares en la población pediátrica. Pacientes y métodos. Se analizó retrospectivamente una base de datos de un laboratorio terciario de neuropatología para identificar a pacientes (mayores de 18 años) sometidos a biopsia muscular entre enero de 2015 y agosto de 2019. Se evaluaron los datos demográficos, la presentación clínica, el diagnóstico, el tratamiento y el seguimiento. Resultados. Se incluyó a 106 pacientes, de los que el 52,8% (n = 56) eran varones. La mediana de edad fue de 6 años (rango intercuartílico: 10 años). Los pacientes se dividieron en ocho grupos, según sospecha diagnóstica clínica: miopatías mitocondriales (n = 29), miopatías congénitas (n = 9), miopatías inflamatorias (n = 8), distrofias musculares (n = 7), valores elevados de creatincinasa en el suero (n = 7), miopatías metabólicas (n = 5), otros síntomas neuromusculares (n = 30) y múltiples sospechas clínicas (n = 11). La biopsia fue normal en 50 pacientes. De los restantes, 27 mostraron características diagnósticas específicas, y el 88,9% (n = 24) permitió un diagnóstico definitivo: distrofias musculares (n = 7), miopatías metabólicas (n = 5), miopatías congénitas (n = 4), miopatías inflamatorias (n = 4), miopatías mitocondriales (n = 3) y atrofia muscular espinal (n = 1). La histología llevó a un cambio de tratamiento en cuatro pacientes. La mediana de seguimiento fue de un año (rango intercuartílico: 2 años). Conclusiones. El rendimiento diagnóstico de biopsia fue del 22,6% y fue útil en la orientación diagnóstica o terapéutica en el 35,8%. Las técnicas moleculares llevaron a una disminución de las indicaciones de biopsia muscular, pero ésta sigue siendo una herramienta importante para el diagnóstico de enfermedades neuromusculares.

Apolipoprotein E polymorphism interacts with cigarette smoking in progression of multiple sclerosis.

BACKGROUND AND PURPOSE: The influence of apolipoprotein E (ApoE) polymorphism on clinical severity of multiple sclerosis (MS) is still controversial. Cigarette smoking has been suggested to influence the progression of disability in these patients. In this study, we aimed to investigate whether an interaction of smoking with the ApoE polymorphism influences the progression of disability in MS patients. METHODS: Smoking history from 205 female patients with MS was obtained. Clinical data collected include age at onset, disease duration, annual relapse rate, the Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Severity Score (MSSS). ApoE polymorphism was examined in all patients and stratified according to smoking status and associations with the clinical data investigated. RESULTS: There were no significant associations between cigarette smoking and any of the clinical characteristics in the whole group of patients. In women carrying the ApoE E4 isoform, smokers had a lower EDSS (P = 0.033) and MSSS (P = 0.023) in comparison with non-smokers. CONCLUSION: Our data suggest that in women with MS carrying the ApoE E4 isoform, cigarette smoking may have a protective influence on disease progression and accumulation of disability. These findings need to be confirmed by future large longitudinal studies.

Predictive and Comparative Study Between Clinic Consensus Document for Pancreas Acceptance and Predictive Value of Preprocurement Pancreas Allocation Suitability Score (P-PASS).

The strict selection of pancreas for transplant has forced the development of different documents to select the suitable organ in order to minimize the risks and complications of the transplant. In 2008, Eurotransplant published the Preprocurement Pancreas Allocation Suitability Score (P-PASS) for pretransplant selection. In 2001 the Hospital Clinic of Barcelona developed a Clinical Consensus Document (CCD). OBJECTIVES: We aimed to analyze the predictive decision of the pancreas acceptance to offers received in the hospital, according to the CCD criteria and compare it with the recommended value of suitability for accepting the pancreas according to the P-PASS value. MATERIAL AND METHODS: We performed a retrospective comparative study between the criteria of selection of the CCD for pancreas from 2016-2017 in comparison with the values obtained if the P-PASS had been used: ≤ 17, acceptance criteria and P-PASS; > 17, risk criteria. We defined the organ reported as rejected or accepted. The accepted organ could be procured and transplanted or discarded. RESULTS: With the CCD criteria, 7 more organs were transplanted than if we only applied the potential P-PASS criteria. In contrast, P-PASS would have ruled out an additional 9% of pancreases in relation to CCD criteria. CONCLUSIONS: According our experience, it is difficult to find an adequate prediction model to select pancreas for transplantation. The application of the DCC criteria increases the number of organs valid for transplantation. At present, new criteria should be re-evaluated within multicenter studies.

Relatives' Perception After Tissue Donation Experience: Results of a Postdonation Phone Survey.

INTRODUCTION: Postmortem tissue donation (TD) requires the establishment of strategies for family approach to clearly explain the characteristics of multi-tissue donation. In a tertiary university hospital with a long tradition of tissue generation, we designed a survey to be applied to tissue donor families to evaluate global hospital care, care from Transplant Coordinators (TC), quality and content of information given about TD, experience, and motivations after TD process. METHODOLOGY: A prospective phone survey of 10 multiple-choice items was conducted to all TD relatives that agreed to donate one or more tissues. RESULTS: From the 166 calls made to TD relatives, 75 (45%) were answered: 50 were cornea donors and 25 were multiple-tissues donors. None of the relatives denied participating, the rest were not found. No statistical differences in demographical variables were found between both types of TD. The hospital and TC care perception, the quality of the given information about the processes of TD, the postdonation experiences in terms of procedures, and the impression about body appearance for relatives regardless of the type of donation, corneas or multiple tissues, were evaluated as good or very good for most of the TD relatives. Our study showed that 83% of the family members would agree to donate again; 40% of the relatives were surprised to be offered the option to donate; 10% did not know if they would donate again. Solidarity was the leading reason for TD. CONCLUSION: The relatives' perception of care is a critical component of the quality evaluation of the TD process. The global evaluation results support our strategies for family approach.

Quality Assessment of Postmortem Tissue Donors in a University Hospital: Detection and Generation Tissue Indicators.

BACKGROUND: Defining quality assessment and measurement tools in the area of tissue donation should be considered to be one of the most important strategies for developing health centers. The aim of this project was to identify, define, and analyze a set of indicators to assess the most important steps in the tissue donor detection and generation processes. METHODS: A prospective, descriptive, and comparative study of all potential tissue donors (TDs) detected and generated in a university hospital was performed. All deceased patients after cardiocirculatory death were evaluated in 2015 by the transplant coordinators (TCs). We defined as detection indicators: total deaths, percentage of detection and evaluation, percentage of clinical contraindications, tissue donor potentiality (TDP; corneal or multitissue potentiality), and the functional detection time (FDT); and as generation indicators: generation rate (corneal or multitissue generation), family request time, number of interviewed relatives, and TC experience (y). RESULTS: The detection and evaluation rate was 100% (n = 1,235); tissue clinical contraindications were 57%, and TDP was 43% (n = 528; corneal, 80%; multitissue, 20%). The FDT was 24 ± 30 minutes. The generation rate was 53.4% (n = 282): corneal, 57% (n = 241); and multitissue, 40% (n = 41). Family request time was 10 ± 17 minutes, average number of interviewed relatives was 2.2 ± 1.6, and 35% of TCs had experience in the field for >5 years. CONCLUSIONS: Obtaining indicators for quality assessment in the area of tissue donation is useful in predicting the outcome of the TD process as well as promoting the approach of continuous improvement.

Successful Pancreas Transplantation From a Deceased Donor Intoxicated With Oral Antidiabetic Agent: A Case Report.

Simultaneous kidney pancreas transplantation (SKP) is a common procedure for the patient with long-term type 1 diabetes mellitus (DM) with terminal renal failure. It is unusual to consider the pancreas from a deceased donor who died after an acute intoxication with oral antidiabetic agent (OAA), which would suggest an abnormal functionality of the organ and preclude the potential use of the graft. We present a case of a successful pancreatic transplantation from a donor who died of acute cerebral edema secondary to severe hypoglycemia induced by OAA acute intoxication.

alpha-dystroglycan isoforms are differentially distributed in adult rat retina.

alpha-Dystroglycan (alpha -DG) is a laminin/agrin receptor expressed in skeletal muscle as well as in nervous system and other tissues. Glycosylation of the core protein of alpha-DG is extensive, variable from tissue to tissue, and functionally relevant. To address differential glycosylation of alpha-DG in the retina, we have investigated the distribution of this protein using two different antibodies: 1B7 directed against the core protein of alpha-dystroglycan, and IIH6 directed against a carbohydrate moiety (Ervasti and Campbell [1993] J Cell Biol 122:809-823). Monoclonal antibody 1B7 recognizes a broader band than IIH6, which seems to recognize only a subset of alpha-DG forms in retina. These data reflect the existence of differentially glycosylated isoforms of alpha-DG. Monoclonal antibody 1B7 shows an extensive staining for alpha-DG in the inner limiting membrane as well as in the ganglion cell and inner plexiform layers labeling Müller cell processes, whereas monoclonal antibody IIH6 staining is restricted to the inner limiting membrane and blood vessels. Our data indicate that there are distinct isoforms of alpha-DG that are localized in apposition to basal lamina in the inner limiting membrane and blood vessels or within the parenchyma of the retina along Müller glia. Both isoforms are expressed in a Müller cell line in culture and coimmunoprecipitate with beta-dystroglycan. These data suggest that DGs may participate in organizing synapses and basement membrane assembly in the retina.

Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration.

BACKGROUND: The suitability of the strain Rhodococcus erythropolis ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated. The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114. RESULTS: A spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing. Both were similar as far as biomass yield is concerned. Cell-linked cholesterol oxidase was extracted with Triton X-114, and this detergent was also used for purification and concentration, following temperature-induced detergent phase separation. Triton X-114 was utilized to purify and to concentrate the cell-linked and the extracellular enzyme. Cholesterol oxidase was found mainly in the resulting detergent-rich phase. When Triton X-114 concentration was set to 6% w/v the extracellular, but not the cell-extracted enzyme, underwent a 3.4-fold activation after the phase separation process. This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium. Fermentation yielded 360 U/ml (672 U/ml after activation), 36% of which was extracellular (65% after activation). The Triton X-114 phase separation step yielded 11.6-fold purification and 20.3-fold concentration. CONCLUSIONS: The results of this work may make attractive and cost-effective the implementation of this bacterial strain and this detergent in a purification-based industrial production scheme of commercial cholesterol oxidase.

Cultured Müller cells have high levels of epidermal growth factor receptors.

High levels of epidermal growth factor (EGF)-receptors have been reported in membrane homogenates of bovine retinas, but the biologic function and tissue target of EGF in the retina have not been established fully. Because EGF participation has been suggested in the mechanisms of wound healing and Müller cells undergo changes after retinal injury, the authors studied EGF receptor expression and functional role of this substance in cultured Müller cells. These cells (isolated from normal rats) were tested for the glial cell markers: vimentin, S-100 protein, and carbonic anhydrase C. These markers were found to be positive through all passages used in the experiments. The 125I-EGF binding in Müller cells was highly specific, concentration dependent, and saturable. Compared with 3T3 fibroblasts, Müller cells bound threefold more EGF. Binding kinetics and Scatchard analyses showed the higher level of binding was related to the greater number of receptors on these cells (Müller cells, 2.4 x 10(5) receptors/cell; 3T3 fibroblasts, 7.1 x 10(4) receptors/cell) rather than a change in affinity of the receptors to bind the ligand. Nonlinear-regression analyses suggested the presence of two classes of affinity sites. The high level of EGF-receptor expression in Müller cells was confirmed by western blot analyses that showed increased reactivity of the approximately 170-kilodalton receptor band to a monoclonal anti-EGF receptor antibody. Moreover, EGF treatment of Müller cells resulted in two- to threefold increase in DNA synthesis, as evidenced by 3H-thymidine uptake studies. These findings support a functional role for EGF in Müller cell proliferation in retinal disease.

Microglial cells invade the outer retina as photoreceptors degenerate in Royal College of Surgeons rats.

PURPOSE: Photoreceptor degeneration is accompanied by the invasion of phagocytic cells into the outer retina of Royal College of Surgeons (RCS) rats. Previous studies suggested that these mononuclear phagocytes were blood-borne macrophages and not retinal pigment epithelial cells nor Müller glia. Thus, immunospecific markers were used to identify these cells and to determine their distribution in the dystrophic retina. METHODS: Retinas from RCS and control (RCS-rdy+) rats were processed for immunocytochemistry using antibodies against phosphotyrosine, which labels both microglial cells and peripheral macrophages, and ED2, which labels peripheral macrophages only. As a positive control to demonstrate ED2-labeling of peripheral macrophages that enter the retina during injury, experiments were performed using needle-punctured Long Evans rat retinas. RESULTS: In normal animals, process-bearing, phosphotyrosine-reactive cells were restricted to the inner retinal layers and the outer plexiform layer. In early dystrophic retinas, phosphotyrosine-reactive cells also were observed in the outer retinal layers. The number of phosphotyrosine-labeled cells in the outer retina increased substantially in later stages of dystrophy. ED2-reactive cells were observed in normal or dystrophic retinas only after needle puncture. CONCLUSIONS: These findings suggest that phagocytic cells during the early stages of dystrophy in RCS rat retinas are derived from resident microglial cells, not from peripheral macrophages. The migration of microglial cells into the outer retina when photoreceptor cells begin to degenerate further suggests that they may play a major role in photoreceptor cell death in the dystrophic retina.

Human Müller cells express VEGF183, a novel spliced variant of vascular endothelial growth factor.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor expressed as multiple RNA transcripts due to alternative splicing. During an investigation of the expression of VEGF mRNA in human Müller cells cultured under hypoxic conditions, a cDNA species was isolated whose size was incompatible with known VEGF transcripts. This study was performed to determine the nucleotide sequence of the candidate VEGF species and examine the effects of hypoxia on its expression. METHODS: Cultured human Müller cells were exposed to normoxic (20% O2, 5% CO2, 75% N2) or hypoxic (2% O2, 5% CO2, 93% N2) conditions at 37 degrees C for 4 hours and processed for reverse transcription-polymerase chain reaction (RT-PCR), molecular cloning, Southern hybridization, nucleotide sequencing, semiquantitative RT-PCR, and ribonuclease protection assay. RESULTS: The nucleotide sequence of the novel VEGF species isolated from human Müller cells had a short exon 6-encoded sequence without 18-bp nucleotides immediately upstream of the exon 7-encoded sequence in VEGF189. The 18-bp deletion (corresponding to the six amino acids Tyr-Lys-Ser-Trp-Ser-Val) was compatible with a polypeptide containing 183 amino acids (VEGF183). Although VEGF183 mRNA was found in all tissues studied, its expression seemed to be higher than that of VEGF 189 in the brain and spleen; lower in the kidney, retina, skeletal muscle, and liver; and at similar level in the heart. Exposure to hypoxic conditions for 4 hours promoted increased levels of VEGF mRNA including that of VEGF183. CONCLUSIONS: The expression of the novel isoform VEGF 183 in human Müller cells, its variable tissue expression, and its modulation by hypoxia may provide another pathway for VEGF induction of angiogenesis in the retina.

Enteral vitamin E supplementation inhibits the cytokine response to endotoxin.

OBJECTIVE: To evaluate the effect of short-term, high-dose enteral supplementation of 3 different vitamin E derivatives: free alpha-tocopherol (VE), alpha-tocopherol succinate (VES), and alpha-tocopherol acetate (VEA) on macrophage and monocyte activation. DESIGN: Sprague-Dawley rats (weight, 150-200 g) were assigned to 1 of 5 experimental groups: saline (control), ethanol (control), VES (100 mg/kg), VEA (100 mg/kg), or VE (100 mg/kg). Rats underwent oral gavage once per day for 5 days with 0.5 mL of their assigned solution. All vitamin E derivatives were diluted in 75% ethanol. Rats were then killed and whole-blood and peritoneal macrophages were harvested and stimulated with lipopolysaccharide (10 microg/mL) in vitro. Tumor necrosis factor (TNF) production was measured by enzyme-linked immunosorbent assay. Additional serum samples were analyzed for alpha-tocopherol concentration by high-performance lipid chromatography. RESULTS: Whole-blood TNF production was maximal in the control groups after 3 hours of incubation and began to decline by 6 hours. Supplementation with all 3 vitamin E derivatives resulted in suppression of lipopolysaccharide-induced TNF production at both time points when compared with both ethanol and saline controls (P<.05, analysis of variance [ANOVA]). All 3 vitamin E derivatives also resulted in significant inhibition of lipopolysaccharide-induced TNF production by peritoneal macrophages when compared with their ethanol-carrier control but not with the saline control (P<.05, ANOVA). The degree of TNF suppression correlated directly with serum alpha-tocopherol levels. CONCLUSIONS: Our data demonstrate that a short-term, high-dose enteral supplementation of vitamin E can modulate the monocyte and macrophage response to endotoxin. These data, along with other animal studies showing a protective effect of vitamin E treatment in sepsis and ischemia-reperfusion injury, suggest a potential role for vitamin E supplementation in patients at risk of the systemic inflammatory response syndrome.

Microglia-derived cytotoxic factors. Part I: Inhibition of tumor cell growth in vitro.

The rarity of neoplasms in the adult mammalian retina has led us to hypothesize the presence or increased expression of 'tumor-inhibitory molecules' in the mature differentiated retina. We have begun to investigate the source(s) of these molecules, and the following study describes the inhibitory activity of a soluble microglia-derived cytotoxic factor on the proliferation of C6 cells, a glial tumor cell line. C6 cells were treated for 24, 48, or 72 h with basal medium or basal medium conditioned by retina-derived Muller cells (MCCM) or microglial cells (MGCM) and assayed for cell proliferation and/or cell death using various techniques involving fluorescent probes, lactate dehydrogenase release, or bromodeoxyuridine uptake. C6 cells increased in number from 24 to 72 h following incubation in basal medium or MCCM, but not in MGCM, where the cells rounded up and retracted their processes. The number of dead cells appeared to be the same in all groups at each time point. Similar findings were observed in the presence of 1-10% serum. About 25% of cells treated with basal medium for 72 h were positive for bromodeoxyuridine as compared with < 1% in MGCM-treated cultures. Our studies suggest that retina-derived microglial cells secrete soluble product(s) that inhibit the growth of C6 cells in culture. These molecules may provide protection for the mature retina against the invasion of tumor cells and may prove useful in the treatment of cancer.

Retina-derived microglial cells induce photoreceptor cell death in vitro.

In animals with retinal degeneration, the presence of activated microglial cells in the outer retina during the early stages of injury suggests that they may be involved in the ensuing photoreceptor cell death. In the following study, we investigated the effects of rat retina-derived microglial cells on a photoreceptor cell line (661w) using cell culture techniques. The difficulty of obtaining pure populations of photoreceptor cells necessitated our use of the 661w photoreceptor cells generated from retinas of transgenic mice. 661w Cells were incubated for 24-48 h in basal medium or basal medium conditioned by activated microglial cells (MGCM) or Müller cells (MCCM), and tested for cell death using lactate dehydrogenase (LDH) assay. The induction of apoptosis in the 661w cells by MGCM was investigated using Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and DNA laddering. Treatment of 661w cells with MGCM for 48 h resulted in approximately 73% of cells dead as compared with 19-20% of cells grown in either basal medium or MCCM. Serum supplementation or pretreatment with heat did not abolish the cytotoxicity of MGCM. More TUNEL-positive cells were observed in MGCM-treated cultures as compared with those in basal medium. Bands in multiples of approximately 180 bp formed DNA ladders in MGCM-treated but not in basal medium-treated samples. Our study shows that microglial cells release soluble product(s) that induce degeneration of cultured photoreceptor cells. Moreover, the mechanism of microglia-induced photoreceptor cell death may involve apoptosis similar to that observed in animals with retinal degeneration.

Rat retinal progenitor cells and a retinal pigment epithelial factor.

Retinal progenitor cells were isolated from explants of neonatal rat retinas and characterized by transmission electron microscopy and reverse transcriptase-polymerase chain reaction and by their response to an isolated retinal pigment epithelial cell cell factor. The isolated progenitor cells demonstrated nuclei with abundant euchromatin typical of progenitor cells and showed the presence of nestin and opsin message. A protein ( approximately 67 kDa) isolated from conditioned media of cultured rat RPE cells promoted the survival of isolated retinal progenitor cells.

RPE secreted proteins and antibody influence photoreceptor cell survival and maturation.

Proteins in media conditioned by retinal pigment epithelial cells (RPE-CM) and an antibody against these proteins (RPE-SP) were tested for their respective effects on rat retinal development in vitro and in vivo. Proteins of RPE-CM were separated in denaturing gels and evaluated by Western blot analysis. Retinal explants from postnatal day 2 (P2) rats were cultured in RPE-CM only or CM diluted with the RPE-SP antibody and, after 7 days, the explants were dissociated into single cells that were immunostained for opsin. RPE-CM or antibody was also injected into the vitreous of postnatal day 7 (P7) Long-Evans rats and analyzed 7 and 21 days later. Electrophoretic analysis of RPE-CM predominantly showed 60-70 kDa proteins; when these proteins were probed with RPE-SP antibody by Western blot, immunoreactive proteins were restricted to this narrow molecular weight range. In P2 retinal explant cultures supplemented with RPE-CM, long ganglion cell-like neurites were detected in 3 days. This activity was nullified in explant cultures grown in RPE-CM titrated with antibody, and these explants appeared to degenerate within 5 days. Over 80% of dissociated retinal cells from explants 7 days after treatment with RPE-CM expressed opsin, compared to only 20% of cells from explants grown in defined medium or serum. Retinas of P14 rats injected intravitreally with RPE-CM at P7 had increased numbers of ectopic photoreceptor cells within the inner nuclear layer when compared to retinas of sham-injected eyes. In contrast, retinas of eyes injected intravitreally with RPE-SP antibody exhibited shorter outer (OS) and inner (IS) segments and thinner outer nuclear (ONL) and outer plexiform (OPL) layers than retinas of sham-injected eyes. In conclusion, proteins in RPE-CM appeared to accelerate and maximize the development of rat photoreceptor cells in vitro, while intravitreal injections of its antibody caused an apparent retardation of outer segment maturation. These results suggest that a protein(s) secreted by RPE plays a key role in normal retinal development, particularly in photoreceptor cell survival and outer segment maturation.

Isolation and culture of retinal microglia.

In the Royal College of Surgeons rat, the migration of phagocytic cells into the subretinal space accompanies photoreceptor cell death during the early stages of retinal dystrophy. These are followed closely by cellular alterations in the retinal pigment epithelium, Müller cells, and outer retinal vessels. We have identified the phagocytic cells as microglia and hypothesized that they may be involved in the above cellular changes. Thus, we developed procedures for their isolation and growth. Our study shows that retina-derived microglia (1) are positive for microglial markers Griffonia simplicifolia isolectin B4, Mac-1 alpha, phosphotyrosine, and vimentin; (2) are highly phagocytic; and (3) respond to macrophage colony stimulating factor by proliferating. This culture system would provide a valuable tool in studying mechanisms of cellular alterations in retinal disease.

Increased vascular density and vitreo-retinal membranes accompany vascularization of the pigment epithelium in the dystrophic rat retina.

Observations of vascularization of the retinal pigment epithelium (RPE) and formation of vitreo-retinal membranes (VRMs) in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy suggest that vascular proliferation occurs in this model. To test this hypothesis, we studied the progression of vascular changes in RCS and age-matched control rats using quantitative light microscope morphometry and electron microscopy. At 2 weeks, prior to photoreceptor degeneration, the dystrophic retina is comparable with the control. By 2 months, extensive degeneration of photoreceptor cells results in significant thinning of the dystrophic retina as compared with the control. Signs of vascular degeneration are evident at the electron microscope level--"ghost" vessels consisting of acellular basal lamina surrounded by amorphous electron-dense material; degenerating endothelial cells and pericytes; and abnormal deposits of extracellular matrix (ECM) material around blood vessels. Vascular degeneration is accompanied by glial changes in the form of necrotic perivascular glial processes and abnormal ECM deposits among the altered Muller cell processes. At 2-4 months in the dystrophic retina, numbers of vessel profiles in dystrophic retinas are decreased as compared with controls. However, vascular degeneration is overshadowed by the formation of numerous capillary tufts within the RPE layer, which together with retinal thinning results in increased vessel density. Between 4-12 months, the retinal thickness diminishes further, vascularization of the RPE increases, vitreo-retinal membranes are formed, and vascular density increases. In summary, following an initial period of vascular degeneration, vascularization of the RPE is accompanied by an increase in retinal vessel density and by the formation of vitreo-retinal membranes.