A workflow for 3D-CLEM investigating liver tissue.

Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultrastructural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. In addition, combining volume-EM with high-resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high-resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultrastructural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. LAY DESCRIPTION: As cells and tissues exist in three dimensions, microscopy techniques have been developed to image samples, in 3D, at the highest possible detail. In light microscopy, fluorescent probes are used to identify specific proteins or structures either in live samples, (providing dynamic information), or in fixed slices of tissue. A disadvantage of fluorescence microscopy is that only the labeled proteins/structures are visible, while their cellular context remains hidden. Electron microscopy is able to image biological samples at high resolution and has the advantage that all structures in the tissue are visible at nanometer (10-9 m) resolution. Disadvantages of this technique are that it is more difficult to label a single structure and that the samples must be imaged under high vacuum, so biological samples need to be fixed and embedded in a plastic resin to stay as close to their natural state as possible inside the microscope. Correlative Light and Electron Microscopy aims to combine the advantages of both light and electron microscopy on the same sample. This results in datasets where fluorescent labels can be combined with the high-resolution contextual information provided by the electron microscope. In this study we present a workflow to guide a tissue sample from the light microscope to the electron microscope and image the ultra-structure of a specific cell type in the liver. In particular we focus on the incorporation of fiducial markers during the sample preparation to help navigate through the tissue in 3D in both microscopes. One sample is followed throughout the workflow to visualize the important steps in the process, showing the final result; a dataset combining fluorescent labels with ultra-structural detail.

Developing 3D SEM in a broad biological context.

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.

A novel RIPK4-IRF6 connection is required to prevent epithelial fusions characteristic for popliteal pterygium syndromes.

Receptor-interacting protein kinase 4 (RIPK4)-deficient mice have epidermal defects and fusion of all external orifices. These are similar to Bartsocas-Papas syndrome and popliteal pterygium syndrome (PPS) in humans, for which causative mutations have been documented in the RIPK4 and IRF6 (interferon regulatory factor 6) gene, respectively. Although genetically distinct, these syndromes share the anomalies of marked pterygia, syndactyly, clefting and hypoplastic genitalia. Despite the strong resemblance of these two syndromes, no molecular connection between the transcription factor IRF6 and the kinase RIPK4 was known and the mechanism underlying the phenotype was unclear. Here we describe that RIPK4 deficiency in mice causes epithelial fusions associated with abnormal periderm development and aberrant ectopic localization of E-cadherin on the apical membrane of the outer peridermal cell layers. In Xenopus, RIPK4 depletion causes the absence of ectodermal epiboly and concomitant gastrulation defects that phenocopy ectopic expression of dominant-negative IRF6. We found that IRF6 controls RIPK4 expression and that wild-type, but not kinase-dead, RIPK4 can complement the gastrulation defect in Xenopus caused by IRF6 malfunctioning. In contrast to the mouse, we observed only minor effects on cadherin membrane expression in Xenopus RIPK4 morphants. However, gastrulation defects were associated with a virtual absence of cortical actin in the ectodermal cells that face the blastocoel cavity and this was phenocopied in embryos expressing dominant-negative IRF6. A role for RIPK4 in actin cytoskeleton organization was also revealed in mouse epidermis and in human epithelial HaCaT cells. In conclusion, we showed that in mice RIPK4 is implicated in cortical actin organization and in E-cadherin localization or function, which can explain the characteristic epithelial fusions observed in PPSs. In addition, we provide a novel molecular link between IRF6 and RIPK4 that unifies the different PPSs to a common molecular pathway.

Pharmacology of capsaicin-, anandamide-, and N-arachidonoyl-dopamine-evoked cell death in a homogeneous transient receptor potential vanilloid subtype 1 receptor population.

BACKGROUND: Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a primary pain-sensing relay at peripheral sensory nerve endings and is also widespread in the brain, where it is implicated in neurodegeneration. Previous studies of TRPV1 neurotoxicity have utilized heterogeneous receptor populations, non-selective ligands, or non-neuronal cell types. Here, we explored the pharmacology of TRPV1-induced cytotoxicity in a homogeneous, neurone-like cellular environment. METHODS: Cell death was examined in a human neurone-like cell line, stably expressing recombinant human TRPV1. Cytotoxicity was quantified in terms of nuclear morphology and mitochondrial complex II activity. Immunocytochemical markers of apoptotic cell death were also examined. RESULTS: The TRPV1-selective agonist capsaicin, and the endovanilloids anandamide and N-arachidonoyl-dopamine (NADA), induced TRPV1-dependent delayed cell death in a concentration- and time-dependent manner. Capsaicin exposure time was significantly correlated with potency (r(2)=0.91, P=0.01). Release of cytochrome c from mitochondria, activation of caspase-3, and condensed nuclear chromatin were evident 6 h after capsaicin exposure, but cytotoxicity was unaffected by a pan-caspase inhibitor (zVAD-fmk, 50 microM). CONCLUSIONS: We conclude that capsaicin, anandamide, and NADA can initiate TRPV1-dependent delayed cell death in neurone-like cells. This is an apoptosis-like process, but independent of caspase activity.

Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features.

Necroptosis, necrosis and secondary necrosis following apoptosis represent different modes of cell death that eventually result in similar cellular morphology including rounding of the cell, cytoplasmic swelling, rupture of the plasma membrane and spilling of the intracellular content. Subcellular events during tumor necrosis factor (TNF)-induced necroptosis, H(2)O(2)-induced necrosis and anti-Fas-induced secondary necrosis were studied using high-resolution time-lapse microscopy. The cellular disintegration phase of the three types of necrosis is characterized by an identical sequence of subcellular events, including oxidative burst, mitochondrial membrane hyperpolarization, lysosomal membrane permeabilization and plasma membrane permeabilization, although with different kinetics. H(2)O(2)-induced necrosis starts immediately by lysosomal permeabilization. In contrast, during TNF-mediated necroptosis and anti-Fas-induced secondary necrosis, this is a late event preceded by a defined signaling phase. TNF-induced necroptosis depends on receptor-interacting protein-1 kinase, mitochondrial complex I and cytosolic phospholipase A(2) activities, whereas H(2)O(2)-induced necrosis requires iron-dependent Fenton reactions.

Influence of oestradiol and tamoxifen on oestrogen receptors-alpha and -beta protein degradation and non-genomic signalling pathways in uterine and breast carcinoma cells.

Tamoxifen acts as an oestrogen antagonist in the breast reducing cell proliferation, but in the uterus as an oestrogen agonist resulting in increased cell proliferation. Tamoxifen exerts its tissue-specific effects through the oestrogen receptors (ERalpha or ERbeta). The levels and functions of the two ERs affect the response of the target tissue to oestrogen and tamoxifen. We examined the control of ER stability in breast and uterine cell lines using western blotting and RT-PCR. In MCF-7 breast-derived cells, ERalpha and ERbeta proteins were rapidly degraded via the proteasome pathway in response to oestradiol; conversely tamoxifen stabilised both receptors. In Ishikawa uterine-derived cells, oestradiol and tamoxifen stabilised ERalpha but led to degradation of ERbeta by the proteasome pathway. Further investigations showed that oestradiol induced activation of the non-genomic ERalpha/Akt signalling pathway in MCF-7 cells. We have demonstrated that the alternative Erk signalling pathway is activated in Ishikawa cells following oestradiol treatment in the absence of an active proteasome pathway and therefore increased levels of ERbeta. In conclusion, our data have demonstrated tamoxifen or oestradiol control of ER subtype stability and that non-genomic activation of transcription pathways is cell specific.

The dynamics of blood-brain barrier breakdown in an experimental model of glial cell degeneration.

This study was undertaken to investigate the dynamics of blood-brain barrier breakdown in an in vivo rat model of selective CNS vulnerability. 1,3-Dinitrobenzene was used to induce rapid glial degeneration in highly defined areas of the brainstem. Leakage of fluorescent dextran was used to demonstrate the breakdown of the blood-brain barrier, and antibodies to glial and neuronal specific proteins to assess the accompanying cell changes. Beginning 18 h after a toxic dose of dinitrobenzene and before loss of glial ensheathment, a sub-population of blood vessels became permeable to fluorescent dextrans below 500,000 mol. wt in size. By 24h most macroglial cells had been lost from within susceptible areas and vascular leakage had reached peak levels. Macrophage invasion was detected three days following dinitrobenzene. Vessels continued to leak up to four days after the lesion was formed, but by six days blood-brain barrier integrity was largely re-established. Multiple tracer injections over time demonstrated that a single sub-population of vessels was leaking during the experimental period. From these findings we conclude that blood-brain barrier breakdown in this model system is highly selective, graded in extent and molecular weight specificity and not a direct consequence of astrocyte degeneration or microglial activation. This system could be useful in modeling human CNS pathological processes with a vascular component and for understanding in vivo glial blood-brain barrier interactions.

Regional distribution of amyloid-Bri deposition and its association with neurofibrillary degeneration in familial British dementia.

Familial British dementia (FBD), pathologically characterized by cerebral amyloid angiopathy (CAA), amyloid plaques, and neurofibrillary degeneration, is associated with a stop codon mutation in the BRI gene resulting in the production of an amyloidogenic fragment, amyloid-Bri (ABri). The aim of this study was to assess the distribution of ABri fibrillar and nonfibrillar lesions and their relationship to neurofibrillary pathology, astroglial and microglial response using immunohistochemistry, confocal microscopy, and immunoelectron microscopy in five cases of FBD. Abnormal tau was studied with immunoblotting. We present evidence that ABri is deposited throughout the central nervous system in blood vessels and parenchyma where both amyloid (fibrillar) and pre-amyloid (nonfibrillar) lesions are formed. Ultrastructurally amyloid lesions appear as bundles of fibrils recognized by an antibody raised against ABri, whereas Thioflavin S-negative diffuse deposits consist of amorphous electron-dense material with sparse, dispersed fibrils. In contrast to nonfibrillar lesions, fibrillar ABri is associated with a marked astrocytic and microglial response. Neurofibrillary tangles and neuropil threads occurring mainly in limbic structures, are found in areas affected by all types of ABri lesions whereas abnormal neurites are present around amyloid lesions. Immunoblotting for tau revealed a triplet electrophoretic migration pattern. Our observations confirm a close link between ABri deposition and neurodegeneration in FBD.

Transforming growth factor beta in experimentally detached retina and periretinal membranes.

This study was undertaken to determine whether experimental retinal detachment produces changes in retinal localization of three isoforms of transforming growth factor beta (TGF-beta) and the type II receptor for this protein. Neural retinas of young adult cats were detached from the pigment epithelium. Survival times varied from 3 to 28 days to study the temporal course of TGF-beta localization during retinal degeneration. ELISA assay for TGF-beta1 and -beta2 was performed on samples of fluid from the vitreous chamber to determine whether active or inactive TGF-beta was present. Confocal microscopy was used to localize TGF-beta1, -beta2 and -beta3 and the type II TGF-beta receptor at the various detachment durations. Following experimental retinal detachment the levels of TGF-beta2 increased in the vitreous chamber but no changes in TGF-beta1 were detected. Levels were increased 3 days post-detachment and continued throughout the 28 day period studied. The most prominent changes in immunolocalization occurred in the TGF-beta1 and -beta2 isoforms. Increased immunolabeling was seen in Müller cells and ganglion cell bodies. Hypertrophied Müller cell processes formed periretinal membranes that were heavily labeled by the TGF-beta2 antibody. Some increased immunostaining for TGF-beta3 was observed in the ganglion cell bodies. Labeling for the TGF-beta type II receptor was seen in Müller cells, ganglion cells and the inner and outer plexiform layers in both normal and detached retinas. Changes in localization of the receptor after detachment paralleled the changes seen in TGF-beta protein localization. These results demonstrate that retinal detachment induces the synthesis and secretion of TGF-beta2. Growth factor and receptor immunolabeling were increased in Müller cells suggesting that this isoform is involved in the retinal gliotic response and may contribute to the development of proliferative vitreoretinopathy.

F-actin fiber distribution in glomerular cells: structural and functional implications.

BACKGROUND: Glomerular distention is associated with cellular mechanical strain. In addition, glomerular distention/contraction is assumed to influence the filtration rate through changes in filtration surface area. A contractile cytoskeleton in podocytes and mesangial cells, formed by F-actin-containing stress fibers, maintains structural integrity and modulates glomerular expansion. In this study, the glomerular cell distribution of F-actin and vimentin filaments was studied in normal control and nine-month streptozotocin-diabetic rats. Results in situ were compared with observations in tissue culture. METHODS: Microdissected rat glomeruli were perfused to obtain a physiological 25% glomerular expansion over the basal value. Fixation was done without change in glomerular volume. Dual fluorescent labeling of F-actin and vimentin was carried out, and samples were examined by confocal laser scanning microscopy. RESULTS: The podocyte cell bodies and their cytoplasmic projections, including the foot processes, contained bundles of vimentin-containing fibers. Except for a thin layer at the base of foot processes, they did not demonstrate F-actin. While mesangial cells in culture had F-actin as long stress fibers resembling tense cables, mesangial cells stretched in situ contained a maze of short tortuous F-actin fibers organized in bundles that often encircled vascular spaces. This fibrillar organization was disrupted in diabetic glomeruli. CONCLUSION: Mesangial cells, but not podocytes, contain a cytoskeleton capable of contraction that is disorganized in long-term diabetes. Together with previous observations, the distribution of this cytoskeleton suggests that mesangial cell contraction may be involved in the redistribution of glomerular capillary blood flow, but not substantially in the modulation of glomerular distention. Disorganization of stress fibers may be a cause of hyperfiltration in diabetes.

TGF-beta receptor expression and binding in rat mesangial cells: modulation by glucose and cyclic mechanical strain.

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a causal factor in experimental glomerulosclerosis, and it mediates the increased extracellular matrix (ECM) accumulation that occurs in cultured mesangial cells (MCs) exposed to high glucose concentrations and cyclic mechanical strain. This change is associated with increased levels of TGF-beta, but may also involve alterations in receptor expression and binding. METHODS: Rat MCs cultured in media containing either 8 or 35 mM glucose were seeded into culture plates with elastin-coated flexible bottoms. Thereafter, they were subjected to cyclic stretch or static conditions and then examined for 125I-TGF-beta1 binding and expression of TGF-beta receptors at the gene and protein levels. RESULTS: Kinetic studies showed that MCs bound TGF-beta1 in a time- and concentration-dependent manner, expressing 6800 high-affinity receptors per cell, with an apparent dissociation constant (Kd) of 15.4 pM, while cross-linking analysis identified three TGF-beta receptors (betaR) corresponding to betaRI, betaRII, and betaRIII of 54, 73, and 200 kDa, respectively. Immunocytochemical studies of betaRI and betaRII protein revealed MC expression in a homogeneous, punctate distribution, whereas Northern analysis demonstrated the presence of the corresponding mRNAs. Exposure to cyclic stretching significantly increased (10%) the overall number of TGF-beta receptors, whereas ligands associated with betaRs I, II, and III also increased (25 to 50%). The finding of increased (30 to 40%) betaRI and betaRII transcript levels and immunoreactive protein (163 and 59%, respectively) in the absence of significant changes in the apparent Kd indicated that stretch-induced binding was the result of increased receptor synthesis and expression and not due to a change in binding affinity. In a similar, but more dramatic fashion, exposure to high glucose also elevated (50%) the receptor number, as well as the amount of ligands associated with betaRs I, II, and III (100 to 250%). This same treatment also increased the levels of betaRI and betaRII mRNA (30 to 40%) and the immunoreactive protein (82 and 82%, respectively), without significantly altering the binding affinity of the receptor. A concerted or synergistic effect of both stimuli was not evidenced. CONCLUSION: These results suggest that the modulation of TGF-beta receptors may be an additional control point in mediating the glucose- and mechanical force-induced increase in ECM deposition by MCs.

Effects of the neurotrophin brain-derived neurotrophic factor in an experimental model of retinal detachment.

PURPOSE: To examine the effects of brain-derived neurotrophic factor (BDNF) in an animal model of retinal detachment. METHODS: Cat retinas were detached from the retinal pigment epithelium for either 7 or 28 days. Animals received either an intravitreal injection of BDNF (100 ILg) or phosphate-buffered saline (PBS), the vehicle for BDNF. Retinas were evaluated using morphology and immunocytochemistry. The width of the outer segment zone was measured, and the retinas were evaluated for changes in protein expression by labeling with antibodies to rod opsin, phosducin, synaptophysin, calbindin D, and glial fibrillary acidic protein (GFAP). The effect of BDNF on both proliferation and apoptotic cell death was examined. RESULTS: Although there was variability in the treated retinas, most of the animals receiving BDNF had well-organized outer segments that were longer than those in vehicle-treated controls. Immunocytochemistry revealed that treated retinas had consistently less opsin redistribution to the plasma membrane, less phosducin upregulation, and fewer calbindin D-labeled horizontal cell processes. BDNF did not reduce overall cell death in the detachments or death of photoreceptors by apoptosis. However, it significantly reduced the proliferative response of Miller cells and the extent of upregulation of GFAP. CONCLUSIONS. The results suggest that BDNF may aid in the recovery of the retina after reattachment by maintaining the surviving photoreceptor cells, by reducing the gliotic effects in Müller cells, and perhaps by promoting outer segment regeneration.

Distribution of transforming growth factor-beta isoforms in the mammalian retina.

The distribution of transforming growth factor-beta (TGF-beta) was examined in the posterior segment of the monkey, human, and feline eye using antisera to TGF-beta 1, TGF-beta 2, or TGF-beta 3. A number of different antibodies, tissue processing methods, immunolocalization techniques, and microscopic imaging systems were used in an attempt to gain a more comprehensive picture of TGF-beta isoform distribution in the retina and retinal pigmented epithelium (RPE). The results are generally consistent in identifying one or more of the three TGF-beta isoforms in the cytoplasm of a small, overlapping subset of cells. RPE cells, photoreceptors, Mueller cells, ganglion cells, hyalocytes, and cells associated with choroidal and retinal vessels are all represented in this immunoreactive population. No evidence of extracellular labeling was noted. The intracellular distribution of the three isoforms is distinctly different in photoreceptors. Anti-TGF-beta 1 precursor and anti-TGF-beta 2 immunoreactivity is confined primarily to rod outer segments, whereas anti-TGF-beta 3 immunoreactivities are restricted to mitochondria within inner segments. In the RPE, clusters of anti-TGF-beta 2 positive cytoplasmic granules are located near the cells' lateral borders, whereas anti-TGF-beta 3 labeling is concentrated apically. These results provide baseline information from which new hypotheses regarding the function(s) of TGF-beta isoforms in the retina can be formulated.

Transforming growth factor beta 2 is the predominant isoform in the neural retina, retinal pigment epithelium-choroid and vitreous of the monkey eye.

Several techniques were utilized to assess the levels, disposition and cellular sources of isoforms 1 and 2 of transforming growth factor beta (TGF-beta) in the posterior pole of the monkey eye. Freshly dissected tissues, as well as the saline vehicles in which dissections were performed, were analysed by sandwich enzyme-linked immunosorbent assay. In all tissues TGF-beta 2 was the predominant isoform, with beta 2:beta 1 ratios of 6:1 for neural retina (as ng g-1) and 425:1 for vitreous (as pmol l-1). Retinal pigment epithelium (RPE)-Bruch's membrane-choroid complex contained approximately 10 times the amount of both TGF-beta isoforms as neural retina. For first passage cultures of monkey RPE, TGF-beta 2, but not TGF-beta 1, accumulated over time in conditioned media samples. Immunoreactivity for TGF-beta 2 was detected both in tissue sections of posterior pole, specifically in rod outer segments and RPE, and also in the first passage cultures of RPE. Antibodies to specific peptide sequences of both isoforms localized TGF-beta to the outer segments of rod photoreceptors. The apparent sequestration of TGF-beta 2 in photoreceptor outer segments, as well as the in vitro evidence for possible synthesis and release by RPE, suggest that TGF-beta 2 is an important modulator of visual function acting at the retina-RPE interface.

Rapid changes in the expression of glial cell proteins caused by experimental retinal detachment.

We examined the expression of several proteins normally present in Müller's glia after the production of experimental retinal detachment in adult cats. Retinas were detached for one-half to seven days, after which the tissue was processed for correlative immunocytochemistry and biochemistry. Previous studies demonstrated that the intermediate filament proteins glial fibrillary acidic protein and vimentin, increase after long-term retinal detachment (30 to 60 days), whereas glutamine synthetase, carbonic anhydrase C, and cellular retinaldehyde-binding protein all decrease to barely detectable levels. Alterations in Müller cell protein expression are rapid and specific events that can be detected as early as two days after retinal detachment. By seven days, levels of protein expression are similar to those in the long-term retinal detachments. Within the first week after injury the Müller cell processes hypertrophy and begin forming glial scars, which indicates that early intervention may be required to halt or reverse the effects of detachment.

Recovery of photoreceptor outer segment length and analysis of membrane assembly rates in regenerating primate photoreceptor outer segments.

PURPOSE: Photoreceptor outer segments are in a dynamic state of membrane addition and disposal. This study was undertaken to determine how a standardized period of retinal detachment and varying periods of reattachment affect the renewal process. METHODS: To investigate the effects that retinal detachment and reattachment may have on this process, the neural retina from 12 adult rhesus monkeys (Macaca mulatta) was detached from the overlying retinal pigment epithelium (RPE) by subretinal injection of a balanced salt solution. After a standardized detachment period of 7 days, the two tissue layers were reapposed. Animals were labeled with 3H-fucose and killed at times ranging from 3-150 days after reattachment. RESULTS: During the 7 day detachment period, the majority of rod outer segments (ROS) and cone outer segments (COS) degenerated, but inner segments remained intact. During the first week after reattachment, a rapid increase in rod and cone outer segment length occurred in the absence of disc shedding. This was accompanied by re-establishment of a modified morphologic relationship between the apical processes of the RPE and the regenerating outer segments. ROS and COS regained approximately 40% of their control lengths after a 2 wk reattachment period. By 30 days of reattachment, ROS had regained 72% of their normal length and COS had regained approximately 48%. After 150 days of reattachment, photoreceptor outer segment mean length was not statistically different from control areas. Autoradiographic results confirmed that new disc membranes were synthesized after reattachment. The rate of ROS membrane assembly was subnormal at reattachment time points up to 30 days. CONCLUSIONS: Retinal detachment leads to a reduction in photoreceptor outer segment absolute length and membrane assembly rates. Increasing time of retinal reattachment is positively correlated with an increase in outer segment absolute length and a corresponding increase in membrane assembly rates. This recovery pattern in eyes without underlying pathology and after a relatively brief detachment interval may represent the upper limit of the recovery process.

Basic fibroblast growth factor: a potential regulator of proliferation and intermediate filament expression in the retina.

Proliferation of astrocytes, and a concomitant increase of intermediate filaments in astrocytes are two fundamental responses of the CNS to injury. We have previously identified these two events in the retina's response to detachment of the neural retina from the adjoining monolayer of retinal pigmented epithelium. In order to analyze the potential role of basic fibroblast growth factor (bFGF) in these responses, we studied cellular proliferation and intermediate filament protein expression in the retinas of cats and rabbits 4 d and 4 weeks after a single intravitreal injection of 1 microgram of bFGF. Our results show that bFGF stimulates both of these processes in an otherwise normal eye. The eyes that received bFGF had significantly elevated numbers of 3H-thymidine-labeled Müller cells, astrocytes, vascular cells, retinal pigmented epithelial cells, microglia, and macrophages by comparison to control eyes. This proliferation was apparent at 4 d after the injection of bFGF but not after 4 weeks. In control eyes, antibodies to glial fibrillary acidic protein and vimentin labeled intermediate filaments only in the inner (vitread) portion of the Müller cells, the specialized radial astrocytes that span the width of the retina. In eyes that had been injected with bFGF, almost the entire Müller cell cytoplasm was labeled at 4 d after injection; after 4 weeks, the cytoplasmic labeling intensity had increased significantly. Release or activation of endogenous stores of bFGF after injury or disease may be involved in the control of cellular proliferation and intermediate filament expression in the retina and elsewhere in the CNS.

Changes in intermediate filament immunolabeling occur in response to retinal detachment and reattachment in primates.

The immunolabeling patterns for vimentin and glial fibrillary acidic protein (GFAP) were studied in five rhesus monkeys that had undergone retinal detachment or detachment and reattachment. Anti-vimentin and anti-GFAP labeling intensity increased in Müller cells after 2 days of detachment. Weak anti-vimentin labeling of the basal RPE cytoplasm, which was absent in control tissue, was detected 2 days after detachment. After detachment for 7 days and reattachment for 7 or 14 days, the pattern and extent of intermediate filament (IF) labeling changed. In Müller cells, the labeling, which in controls was restricted to processes near the vitreal border of the retina, was present in Müller processes spanning the entire retina. In retinal pigment epithelium (RPE) cells, prominent anti-vimentin labeling was identified in the basal and basolateral cytoplasm. The extent of RPE and Müller cell IF labeling in two animals whose retinas had been detached and then reattached for 150 days was different from that found at either the 7- or 14-day reattachment time points. This suggests that the abnormal IF distribution triggered by detachment may be attenuated after a lengthy period of reattachment.

Immunocytochemical identification of Müller's glia as a component of human epiretinal membranes.

Sections of seven human epiretinal membranes of diverse pathologic origin were labeled with antibodies to cellular retinaldehyde binding protein (CRALBP) and two intermediate filament proteins: glial fibrillary acidic protein (GFAP) and vimentin. All of the membranes studied contained heterogeneous cell populations that exhibited diverse morphologic characteristics. Double labeling with both anti-GFAP and anti-CRALBP positively identified one of the cellular components in these membranes as Müller's glia. In addition, other epiretinal cells exhibited immunolabeling patterns consistent with those found in fibrous astrocytes and retinal pigment epithelial (RPE) cells normally. The results demonstrate that a double-labeling method using CRALBP antibodies, in combination with antibodies to other appropriate antigens, can be used to distinguish between the different epiretinal cell types.

Tritiated uridine labeling of the retina: changes after retinal detachment.

As part of a study designed to examine the response of photoreceptor cells to outer segment injury (retinal detachment), the pattern of RNA labeling ([3H]uridine incorporation) has been determined in detached cat retinas. Retinas were experimentally detached from the adjacent cellular layer (the retinal pigment epithelium:RPE) by injecting fluid into the extracellular space between the retina and RPE. Twenty-four hours before the animals were killed they received intravitreal injections of [3H]uridine. Autoradiograms were prepared from plastic sections 1.0 micron thick taken from detached retinal regions and, because the detachments do not encompass all of the retina, from nearby attached retinal regions. Twenty-four hours after retinal detachment there is a decrease in labeling intensity of the photoreceptors and Müller's glia in the region of detachment (compared to cells in nearby attached regions). Seventy-two hours after retinal separation, the same result is obtained in the photoreceptors, but labeling intensity is greatly increased in both the nuclei and cytoplasm of Müller's glia. The decrease in [3H]uridine labeling of the photoreceptors correlates with a decreased staining intensity of the cytoplasm and ultrastructural signs of necrosis. The striking change in the pattern and intensity of labeling of the Müller cells precedes extensive hypertrophy of these cells and the appearance within their cytoplasm of numerous 10-nm diameter filaments. Two weeks, and also 1 month, after detachment the pattern and labeling levels are similar to those observed 1 day after retinal separation. These data suggest a highly localized change in metabolism because the change in RNA labeling is restricted to the region of detached retina.