Apelin supports primary rat retinal Müller cells under chemical hypoxia and glucose deprivation

Müller cells support the integrity of the blood-retinal barrier, whereas their dysfunction under pathological conditions may contribute to retinal edema formation. The apelin peptide, as the endogenous ligand of G protein-coupled receptor APJ, participates in numbers of physiological and pathological processes. Recent studies highlight its emerging role against ischemic injury. Our study aimed to investigate the potential neuroprotection of apelin for primary rat retinal Müller cells under hypoxia or glucose-deprivation (GD) by cell viability, migration and apoptosis, as well as apelin/APJ immunofluorescence labeling and mRNA expression. The results showed that exogenous apelin significantly stimulated Müller cells viability and migration under normal, hypoxic and glucose-free condition, also prevented apoptosis. Apelin immunoreactivities represented weak and diffuse staining in the cytoplasm, along with restricted nuclear APJ expression. They both appeared stronger immunoreactivities after 12h hypoxia. Under hypoxic stress, apelin mRNA expression began to increase at 6h (9.97 folds, p<0.01), and APJ mRNA also up-regulated (2h 6.50 folds, p<0.05; 4h 2.25 folds, p<0.05; 6h 14 folds, p<0.01), whereas they both down-regulated during 4-12h GD. Our results suggested that apelin induced the tolerance of Müller cells to hypoxia and GD. Its administration might be a promising protection for blood-retinal barrier to ischemia.     

Ultrastructural localization of blood-retinal barrier breakdown in diabetic and galactosemic rats

Breakdown of the blood-retinal barrier (BRB) is an early event in diabetic and galactosemic rats, but the location and nature of the specific defect(s) are controversial. Using an electron microscopic immunocytochemical technique, the retinas of normal, diabetic, and galactosemic rats were immunostained for endogenous albumin. Normal rats showed little evidence of BRB breakdown at either the inner barrier (retinal vasculature) or the outer barrier (retinal pigment epithelium) (RPE). In diabetic and galactosemic rats, as was true in human diabetics, BRB breakdown occurred predominantly at the inner BRB, but in some cases at the outer barrier as well. Treatment with the aldose reductase inhibitor sorbinil largely prevented BRB failure in galactosemic rats. In the inner retina of diabetic and galactosemic rats, albumin was frequently demonstrated on the abluminal side of the retinal capillary endothelium (RCE) in intercellular spaces, basal laminae, pericytes, ganglion cells, astrocytes, and the perinuclear cytoplasm of cells in the inner nuclear layer. Albumin did not appear to cross RCE cell junctions; however, it was occasionally seen in RCE cytoplasm of galactosemic rats. In the outer retina, albumin was frequently detected in the subretinal space, in the intercellular space between photoreceptors, and in the perinuclear cytoplasm of photoreceptor cells, but was only infrequently found in the RPE cells constituting the barrier. Albumin derived from the choroidal vasculature did not appear to cross the tight junctions of the RPE. These findings suggest that specific sites of BRB compromise are infrequent but that once albumin has crossed the RCE or RPE it freely permeates the retinal tissue by filling intercellular spaces and permeating the membranes of cells not implicated in BRB formation. The diffuse cytoplasmic staining of some RCE and RPE cells suggests that the predominant means of BRB breakdown in diabetes and galactosemia involves increased focal permeability of the surface membranes of the RCE and RPE cells rather than defective tight junctions or vesicular transport.     

Vascular permeability (problem of the blood-brain barrier) in the pineal organ of the rainbow trout,Salmo gairdneri

Summary The problem of the blood-brain barrier in the pineal organ of the rainbow trout, Salmo gairdneri, was investigated following intraperitoneal or intracardial injections of several tracers and dyes with different molecular weights. As demonstrated at the light-microscopic level, repeated injections of trypan blue or horseradish peroxidase (HRP) resulted in an accumulation of these substances in the pineal epithelium (parenchyma). By use of the electron microscope, HRP was found in electron-dense bodies, probably lysosomes, in (i) the endothelial cells and perivascular macrophages 4 h after intraperitoneal injection, (ii) the supporting cells and intrapineal or luminal macrophages 8 h after injection, and (iii) the receptor cells 24 h after injection of the tracer. Ferritin particles penetrated the fenestrated endothelium of pineal capillaries. They were confined to vesicles, vacuoles and the smooth endoplasmic reticulum of the supporting cells as well as to the synaptic vesicles and the smooth endoplasmic reticulum of the pineal photoreceptors. The intercellular passage of tannic acid mixed with the fixative was blocked at the luminal junctional complex separating the pineal lumen from the basal portion of the pineal epithelium. The passive intercellular transport of substances with high molecular weight from the bloodstream to the cerebrospinal-fluid compartment is thus prevented. However, no blood-brain barrier exists for exogenously administered proteins, which are rapidly taken up by pineal cells and actively transported in a transcellular manner.The findings on the blood-brain barrier of the pineal organ of the rainbow trout are discussed with particular reference to the endocrine capacity of pineal sensory organs.Fellow of the Alexander von Humboldt Foundation, Federal Republic of Germany.     

Movement of horseradish peroxidase in rabbit submandibular glands after ductal injection

Synopsis Horseradish peroxidase (HRP) has been used as a tracer to study movements of solutions injected retrogradely via the duct of submandibular glands in rabbits. 0.1 ml of solution was injected either manually or by a constant hydrostatic pressure, and the subsequent distribution of HRP in the gland and duct at different times after injection has been examined histochemically at light and electron microscopical levels.Shortly after the injections, strong interstitial staining for peroxidase resulted from passage between acinar cells. Some sites of cellular uptake were observed and staining occurred in some ductal cells even when the duct had been cut at the hilum to minimize pressure effects. It is not known whether this diffuse uptake represents a physiological or pathological phenomenon. Some interstitial activity still remained 24 hr after injection but had disappeared by 48 hr. Inflammatory cells first appeared in the gland about 4 hr after the injection and slowly increased up to about 24 hr after injection.The results indicate that the HRP reaches the interstices of the gland principally by penetration between acinar cells, and that the junctional complexes between striated duct cells appear to be more resistant to disruption by luminal pressures.     

Immunohistochemical localization of blood-retinal barrier breakdown sites associated with post-surgical macular oedema

Summary Post-surgical macular oedema results from blood-retinal barrier breakdown, but it is not accompanied by structural abnormalities in the retinal vessels or retinal pigmented epithelium. Previous studies, using horseradish peroxidase in a primate model, suggested that leakage occurs primarily through this epithelium. This study was conducted to localize sites of the barrier breakdown in humans following different types of intra-ocular surgery and to compare them with eyes affected with ocular inflammatory disease, ocular infection, and choroidal melanoma. Paraffin sections of eyes were immunohistochemically stained for albumin to localize extravascular albumin, which was graded in a masked study. With aphakia/pseudophakia, penetrating keratoplasty, ocular inflammatory disease, ocular infection, and choroidal melanoma, barrier breakdown occurred primarily at the inner blood-retinal barrier (retinal vasculature), but leakage also occurred at the outer barrier (retinal pigmented epithelium). After retinal re-attachment surgery, the inner and outer blood-retinal barriers were equally compromised. Vascular leakage in the optic nerve head coincided with barrier failure in these disorders. The widespread pattern of blood-retinal barrier compromise with leakage at multiple sites suggests that soluble mediators are likely to play a role in postsurgical macular oedema, ocular inflammatory disease, and choroidal melanoma.     

Oxidized low density lipoprotein-induced senescence of retinal pigment epithelial cells is followed by outer blood-retinal barrier dysfunction

Age-related macular degeneration is the most common cause of vision loss in the elderly, which starts from aging processes of retinal pigment epithelial cells. Among variable risk factors in occurrence and progression of age-related macular degeneration, oxidized low density lipoprotein could be causally involved in pathobiological changes of RPE cells. Herein we showed that oxidized low density lipoprotein-induced senescence of retinal pigment epithelial cells is followed by outer blood-retinal barrier dysfunction. Under sub-lethal concentration, oxidized low density lipoprotein could promote advanced senescence of retinal pigment epithelial cells. Interestingly expression of CRALBP and RPE 65, indicators of retinal pigment epithelial cell differentiation, was decreased by oxidized low density lipoprotein. In addition, oxidized low density lipoprotein induced reactive oxygen species production and up-regulated inflammatory factors such as tumor necrosis factor-α and vascular endothelial growth factor, when β-catenin, a critical mediator of the canonical Wnt pathway, was also elevated. Oxidized low density lipoprotein increased paracellular permeability of retinal pigment epithelial cells, when zonula occludens-1 at intercellular junctions markedly decreased as well. Furthermore, in retinal pigment epithelial cells and choriocapillaris of human apolipoprotein E2 transgenic mouse eye, increased vascular endothelial growth factor and decreased zonula occludens-1 expression was observed. Therefore, our results suggest that oxidized low density lipoprotein could promote senescence of retinal pigment epithelial cells which leads to induce outer blood-retinal barrier dysfunction as an early pathogenesis of age-related macular degeneration.     

Electron microscopic immunocytochemical demonstration of blood-retinal barrier breakdown in human diabetics and its association with aldose reductase in retinal vascular endothelium and retinal pigment epithelium

Summary Light-microscopic immunohistochemical staining for albumin has been used to localize sites of blood-retinal barrier (BRB) breakdown in ocular disorders, but the mechanism for BRB compromise cannot be resolved at this level. Using eyes up to 2 days post-mortem from normal patients or from patients with diabetic retinopathy, or other disorders known to cause BRB failure, electron-microscopic immunocytochemistry reveals focal breakdown of the inner BRB, comprised of the retinal vascular endothelium (RVE), which appears to be mediated by diffuse permeation of the RVE cells and by vesicular transport. Permeation of the retinal pigmented epithelial (RPE) cells that comprise the outer BRB also occurs, but there is no evidence of opening of tight junctions between RVE or RPE in any of the disorders evaluated. Increased aldose reductase (AR) expression in the RVE and RPE cells of diabetics as well as in the perivascular retinal astrocytes, which interact with RVE cells to establish the inner BRB, suggests that AR activity and the subsequent intracellular accumulation of sorbitol in these cell types may impair the function of the BRB in diabetes.     

Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin

Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood-brain or blood-retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65- or 2.57-fold increase in content, respectively. A similar two-fold increase in occludin mRNA was observed by real-time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO-1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes.     

Expression of receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin in the inner blood-retinal barrier of rats

The retina is protected from somatic circulation by the blood-retinal barrrier (BRB) composed of tight junctions between retinal vascular endothelial cells (the inner BRB) and those between retinal pigment epithelial cells (the outer BRB). Our recent studies showed that glial cell line-derived neurotrophic factor (GDNF) secreted from astrocytes regulates the permeability of the BBB. In the present study, we immunohistochemically examined the expression of GDNF, neurturin (NTN) and their receptors, GFRalpha1 for GDNF and GFRalpha2 for NTN, because the capillaries of the inner BRB show specialization very similar to the blood-brain barrier (BBB). GDNF and NTN were detected in glial fibrillary acidic protein (GFAP)-positive cells, including Müller cells. GFRalpha1 and GFRalpha2 were localized in von Willebrand factor-positive cells. GDNF and NTN enhanced the barrier function of endothelial cells derived from porcine brain cortex. These results strongly suggest that the barrier function of the BRB is regulated by GDNF and NTN secreted from glial cells, like the BBB.     

The permeability of muscle capillaries to horseradish peroxidase

The main objective of this study was to determine the pathways by which horseradish peroxidase (HRP) can cross the endothelium of muscle capillaries. Specimens of mouse diaphragm were fixed for cytochemical analysis at various intervals after intervenous injection of 0.5 mg HRP, at 4 min after intervenous injection of varied amounts of HRP, and at 4 min after intervenous injections in various volumes of isotonic NaCl. Our findings indicate that endothelial junctions serve as a barrier which may allow passage of very limited amounts of HRP. They also suggest that endothelial vesicles transfer HRP from the capillary lumen to the pericapillary interstitium as well as in the reverse direction. Increasing the volume of solution injected to approximately 30% of total blood volume did not increase the amount of HRP that left the capillary lumen. Our results with HRP do not provide clearcut evidence that endothelial junctions are the site of the small pore.     

Mice lacking insulin or insulin-like growth factor 1 receptors in vascular endothelial cells maintain normal blood-brain barrier

The blood-brain barrier (BBB) is created by a combination of endothelial cells with tight junctions and astrocytes. One of the key tight junction proteins, zona occludens-1 (ZO-1), has been reported to be stimulated in its expression by insulin and IGF-1. To assess the role of insulin and IGF-1 in endothelial cells in the BBB we have utilized mice with a vascular endothelial cell-specific knockout of the insulin receptor (VENIRKO) and IGF-1 receptor (VENIFARKO). Both of these mice show a normal BBB based on no increase in leakage of Evans blue dye in the brain of these mice basally or after cold injury. Furthermore, the structural integrity of the BBB and blood-retinal barrier (BRB) was intact using the vascular markers lectin B-4 and ZO-1, and both proteins were properly co-localized in both brain and retinal vascular tissue of these mice. These observations indicate that neither insulin nor IGF-1 signaling in vascular endothelial cells is required for development and maintenance of BBB or BRB.     

Chemokine Mediated Monocyte Trafficking into the Retina: Role of Inflammation in Alteration of the Blood-Retinal Barrier in Diabetic Retinopathy

Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB). We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2) was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP⁺ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1⁺/CD11b⁺ (monocyte/macrophage and microglia) cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2^−/−) mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.     

The permeability of the primary decidual zone in the rat uterus: an ultrastructural tracer and freeze-fracture study

The rat primary decidual zone (PDZ) is a transitory avascular region of transformed fibroblasts surrounding the implanting embryo. Studies using fluorescein-labeled tracers have shown that the PDZ is selectively permeable to macromolecules, permeability decreasing with increasing molecular weight. In the present study we investigated the morphologic basis of the permeability barrier. Horseradish peroxidase (HRP) or HRP-labeled immunoglobulin G (IgG-HRP) was administered i.v. to rats on Day 7 of pregnancy, and the animals were killed 30 min to 2 h later. The reaction product of HRP was the same density in uterine blood vessels as in the intercellular spaces of the endometrium and PDZ at 30 min and 1 h after administration. Two hours after administration, the reaction product of IgG-HRP was dense in uterine blood vessels, much less dense in the interstitial spaces of the endometrium, and was not detected in the PDZ. There was an abrupt change in the density of the IgG-HRP reaction product at the intercellular clefts between endothelial cells, where cellular junctions were observed in control tissue. This suggests that the passage of large macromolecules from blood to the implantation chamber is limited initially by cellular junctions between capillary endothelial cells. The exclusion of IgG-HRP from the PDZ indicates that an additional barrier(s) to macromolecules in this region. Lanthanum nitrate tracer was uniformly present throughout the intercellular spaces of the PDZ except at tight junctions between decidual cells. Freeze-fracture replicas of the PDZ showed tight junctions that varied from single strands to interconnected networks of strands oriented mainly parallel to the long axis of the PDZ. Some strands were discontinuous. The tight junctions between decidual cells appear to be functionally discontinuous because HRP readily penetrated the PDZ, but such junctions may retard larger macromolecules such as IgG-HRP. The biological significance of the permeability barrier of the PDZ is discussed.     

THE INFLUENCE OF INTRAVASCULAR FLUID VOLUME ON THE PERMEABILITY OF NEWBORN AND ADULT MOUSE LUNGS TO ULTRASTRUCTURAL PROTEIN TRACERS

The permeability of the alveolar-capillary membrane of newborn and adult mice to horseradish peroxidase (HRP) and catalase was studied by means of ultrastructural cytochemistry, and the permeability to ferritin was studied by electron microscopy. The influence of varying volumes of intravenously injected fluid on the rate of leakage of the tracers from pulmonary capillaries was examined. The tracers were injected intravenously and the mice were sacrificed at timed intervals. Experiments on newborn mice with intranasally instilled HRP were also done. The tissues were fixed in formaldehyde-glutaraldehyde fixative. Chopped sections were incubated in Graham and Karnovsky's medium for peroxidase and in a modification of this medium for catalase. Tissues were postfixed in OsO₄ and processed for electron microscopy. In both newborn and adult mice, the ready passage of peroxidase through endothelial clefts was dependent on the injection of the tracer in large volumes of saline. When the tracer was injected in small volumes of saline, its passage through endothelial clefts was greatly reduced. Endothelial junctions of newborn mice were somewhat more permeable to HRP than those of adult mice. In all animals, alveolar epithelial junctions were impermeable to HRP. Catalase and ferritin did not pass through endothelial junctions. Intranasally instilled HRP in newborn mice was taken up by pinocytotic vesicles and tubules of flat alveolar cells.     

Cancer-associated retinopathy: A new mechanistic insight on vascular remodeling

Cancer-associated retinopathy (CAR) is believed to be associated with autoimmune responses against retinal specific antigens. However, CAR patients often show little evidence of immunological reactions at the cellular or molecular levels in their eyes. We have recently shown that tumor-derived VEGF and PlGF significantly remodel the retinal vasculature by ablation of pericytes and impair the blood-retinal barrier, leading to increased vascular leakage. Surprisingly, VEGFR1, but not VEGFR2, is the primary receptor that transduces signals in endothelial or mural cells to produce these vascular pathologies. These results demonstrate that tumor-derived angiogenic factors significantly confer the development of CAR and anti-VEGF agents might be potentially used for the treatment of CAR.     

Environmental Enrichment Protects the Retina from Early Diabetic Damage in Adult Rats

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Available treatments are not completely effective. We analyzed the effect of environmental enrichment on retinal damage induced by experimental diabetes in adult Wistar rats. Diabetes was induced by an intraperitoneal injection of streptozotocin. Three days after vehicle or streptozotocin injection, animals were housed in enriched environment or remained in a standard environment. Retinal function (electroretinogram, and oscillatory potentials), retinal morphology, blood-retinal barrier integrity, synaptophysin, astrocyte and Müller cell glial fibrillary acidic protein, vascular endothelial growth factor, tumor necrosis factor-α, and brain-derived neurotrophic factor levels, as well as lipid peroxidation were assessed in retina from diabetic animals housed in standard or enriched environment. Environmental enrichment preserved scotopic electroretinogram a-wave, b-wave and oscillatory potential amplitude, avoided albumin-Evan''s blue leakage, prevented the decrease in retinal synaptophysin and astrocyte glial fibrillary acidic protein levels, the increase in Müller cell glial fibrillary acidic protein, vascular endothelial growth factor and tumor necrosis factor-α levels, as well as oxidative stress induced by diabetes. In addition, enriched environment prevented the decrease in retinal brain-derived neurotrophic factor levels induced by experimental diabetes. When environmental enrichment started 7 weeks after diabetes onset, retinal function was significantly preserved. These results indicate that enriched environment could attenuate the early diabetic damage in the retina from adult rats.     

Simultaneous immunohistochemical demonstration of intra-axonally transported markers and neuropeptides in the peripheral nervous system of the guinea pig

Summary Projections and peptide neurotransmitter/neuromodulator content of autonomic and visceral afferent neurons of the guinea pig were studied after application of the subunit B of cholera toxin (CTB) with or without horseradish peroxidase (HRP) as retrograde and anterograde tracers and subsequent immunohistochemical processing for double staining using antibodies raised to CTB, HRP and various neuropeptides. The results demonstrate that substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing dorsal root ganglion cells project to the pylorus as well as to the celiac superior mesenteric and stellate ganglia as demonstrated with both retrograde and anterograde transport methodology. Binding studies revealed that a small number of the CTB-binding dorsal root ganglion cells contains immunoreactivity to SP and CGRP. The majority of the CTB-binding cells is SP- and CGRP-negative and terminate in the deeper parts of the dorsal horn. After injection of CTB conjugated to HRP (B-HRP) into the nodose ganglion, both motor and sensory elements were labeled in the medulla oblongata. Some of the CTB labeled vagal sensory nerve fibers in the nucleus tractus solitarii (NTS) were also found to contain immunoreactivity to SP or CGRP. The tracer was also transported through the peripheral branch of the nodose ganglion cells and labeled terminals in the esophagus.     

Septate junctions are required for ommatidial integrity and blood-eye barrier function in Drosophila

The anatomical organization of the Drosophila ommatidia is achieved by specification and contextual placement of photoreceptors, cone and pigment cells. The photoreceptors must be sealed from high ionic concentrations of the hemolymph by a barrier to allow phototransduction. In vertebrates, a blood-retinal barrier (BRB) is established by tight junctions (TJs) present in the retinal pigment epithelium and endothelial membrane of the retinal vessels. In Drosophila ommatidia, the junctional organization and barrier formation is poorly understood. Here we report that septate junctions (SJs), the vertebrate analogs of TJs, are present in the adult ommatidia and are formed between and among the cone and pigment cells. We show that the localization of Neurexin IV (Nrx IV), a SJ-specific protein, coincides with the location of SJs in the cone and pigment cells. Somatic mosaic analysis of nrx IV null mutants shows that loss of Nrx IV leads to defects in ommatidial morphology and integrity. nrx IV hypomorphic allelic combinations generated viable adults with defective SJs and displayed a compromised blood-eye barrier (BEB) function. These findings establish that SJs are essential for ommatidial integrity and in creating a BEB around the ion and light sensitive photoreceptors. Our studies may provide clues towards understanding the vertebrate BEB formation and function.     

Simultaneous immunohistochemical demonstration of intra-axonally transported markers and neuropeptides in the peripheral nervous system of the guinea pig

Projections and peptide neurotransmitter/neuromodulator content of autonomic and visceral afferent neurons of the guinea pig were studied after application of the subunit B of cholera toxin (CTB) with or without horseradish peroxidase (HRP) as retrograde and anterograde tracers and subsequent immunohistochemical processing for double staining using antibodies raised to CTB, HRP and various neuropeptides. The results demonstrate that substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing dorsal root ganglion cells project to the pylorus as well as to the celiac superior mesenteric and stellate ganglia as demonstrated with both retrograde and anterograde transport methodology. Binding studies revealed that a small number of the CTB-binding dorsal root ganglion cells contains immunoreactivity to SP and CGRP. The majority of the CTB-binding cells is SP- and CGRP-negative and terminate in the deeper parts of the dorsal horn. After injection of CTB conjugated to HRP (B-HRP) into the nodose ganglion, both motor and sensory elements were labeled in the medulla oblongata. Some of the CTB labeled vagal sensory nerve fibers in the nucleus tractus solitarii (NTS) were also found to contain immunoreactivity to SP or CGRP. The tracer was also transported through the peripheral branch of the nodose ganglion cells and labeled terminals in the esophagus.     

Absorption of intraarticularly injected horseradish peroxidase in synoviocytes of rat synovial membrane: an ultrastructural-cytochemical study

The ability of type A and type S synoviocytes to absorb horseradish peroxidase (HRP) and the intracellular fate of this tracer were studied by electron microscopic cytochemistry. Different concentrations of HRP (0.1-5 mg/ml) were injected into the left knee joint of rats and at intervals ranging from 1 min to 24 hr after injection the synovial membrane was fixed and incubated for HRP. Type A synoviocytes showed a striking ability to absorb HRP at low concentrations. At 1 and 5 min after injection reaction product was localized in coated pits and coated vesicles (110 nm) as well as in smooth-walled vesicles, vacuoles, and tubules. At 15 min to 4 hr postinjection the lysosomal system became increasingly loaded with reaction product. At 24 hr after injection reaction product had disappeared. At higher concentrations of HRP similar observations were made in the A cells, but reaction product was still apparent in lysosomes at 24 hr postinjection. With respect to type S synoviocytes no reaction product was detected within these cells at any time interval after injection of low concentrations of HRP. However, at 5 min after injection of higher concentrations of HRP reaction product was localized in smooth vesicles and vacuoles mainly restricted to the large cytoplasmic processes facing the joint cavity. At 30 min to 4 hr postinjection the lysosomal system became progressively more loaded with HRP reaction product. At 24 hr after injection reaction product still remained in the lysosomal system. The present findings that type A and type S synoviocytes showed major differences with respect to endocytic capacity and cellular structures involved in absorption of HRP support the interpretation that the A and S cells represent two distinct types of cells and further suggest that endocytosis in these two types of cells serve different functions.