Presence and localization of neurotrophins and neurotrophin receptors in rat lacrimal gland

PURPOSE: To determine which of the neurotrophins (NTs)-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4)-and their receptors (NTrs), TrkA, TrkB, TrkC, and p75, are present in the adult rat lacrimal gland. METHODS: RT-PCR was performed on RNA isolated from male rat lacrimal gland, using oligonucleotides specific to each NT and NTr. The presence of NT and NTr protein, was determined by Western blot analysis of lacrimal gland homogenate or membranes. The location of NTs and NTrs was determined by immunofluorescence histochemistry. Western blot analyses and immunofluorescence microscopy were performed using primary rabbit polyclonal antibodies raised against NTs and NTrs. RESULTS: RT-PCR showed positive bands at the appropriate sizes for NGF, BDNF, NT3, and NT4, and for the receptors TrkA, TrkB, TrkC, and p75. Western blot analysis confirmed these results, showing that the lacrimal gland expresses NGF, BDNF, NT3, and NT4 as well as the NTrs TrkA, TrkB, and TrkC and the p75 protein. NGF, BDNF, NT3, and NT4 were localized in the lacrimal gland acini with differing cellular distributions, whereas TrkA, TrkB, and TrkC, were localized in myoepithelial cell and ductal cell membranes. The protein p75 was expressed only on myoepithelial cell membranes. CONCLUSIONS: Members of the neurotrophin family of growth factors and their receptors are present in rat lacrimal gland, which suggests a role for NTs and their receptors in the lacrimal gland.     

In vivo expression of neurotrophins and neurotrophin receptors is conserved in adult porcine retina in vitro

PURPOSE. To characterize and compare the expression of neurotrophins (NTs) and their receptors within adult porcine retinal ganglion cells (RGCs) in vivo and in vitro. METHODS. The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and -4 (NT-4), and their high-affinity receptors TrkA, TrkB, TrkC and low-affinity receptor p75, was analyzed in adult porcine retinal sections by immunohistochemistry. In addition, adult porcine retinas were dissociated and cultured in four different conditions: control, semipure RGCs, supplemented with BDNF, or seeded on Müller glia feeder layers. Double immunostaining was performed with antibodies to NTs or their receptors combined with neurofilament antibody to identify RGCs in culture. RESULTS. In vivo, immunolabeling of NGF was very faint, BDNF was especially prominent in RGCs and inner nuclear layer cells, NT-3 stained widespread nuclei, and NT-4 was undetectable. TrkA immunoreactivity was visible in the nerve fiber layer, TrkB staining was within RGC bodies, TrkC was undetectable, and p75 was widely expressed across the retina, within the Müller glia. Expression of neurotrophins and their receptors was maintained in all four models of adult RGCs in vitro, showing that expression was not influenced by substrate or culture conditions. We observed prominent staining of TrkA within growth cones, and a clear expression of p75 within a subpopulation of RGCs in vitro. CONCLUSIONS. These findings demonstrate that the expression of NTs and their receptors within adult porcine RGCs is maintained in vitro, under conditions of limited interaction with neighboring neurons and deprived of afferent inputs and target tissue. TrkA may be involved in regeneration of nerve terminals.     

Role of neurotrophins and neurotrophin receptors in rat conjunctival goblet cell secretion and proliferation

PURPOSE: To determine which neurotrophins (NTs)-nerve growth factor (NGF), brain-derived neurotrophin (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4)-and their receptors (NTrs) TrkA, TrkB, TrkC, and p75 are present in rat conjunctiva and cultured rat goblet cells (CGCs) and whether NTs stimulate glycoconjugate secretion or cell proliferation. METHODS: Western blot analysis and immunofluorescence microscopy determined presence and location of NTs and NTrs. CGCs were incubated with NTs (10(-12)-10(-8) M) for 2 or 24 hours to measure secretion or proliferation, respectively. An enzyme-linked lectin assay analyzed glycoconjugate secretion. WST-8 determined cell proliferation. RESULTS: Western blot analysis showed all NTs and NTrs in both conjunctiva and CGCs. The cytoplasm of conjunctival stratified squamous cells and goblet cell lateral membranes contained NGF, BDNF, and NT4. Stratified squamous cell membranes contained NT3. In CGCs, NGF and BDNF had punctuate perinuclear staining. The nucleus contained NT3 and cytosol contained NT4. TrkA, TrkB, and p75 immunoreactivity was on conjunctival goblet cell lateral membranes. Plasma membranes of the basal layer of stratified squamous cells contained TrkA. Stratified squamous cell and goblet cell nuclei contained TrkC. In CGCs, all NTrs were present in the nucleus. NGF and BDNF, but not NT3 and NT4, induced a concentration-dependent stimulation of secretion from CGCs with a maximum increase of 10(-9) M each. No effect on cell proliferation was detected with any NTs. CONCLUSIONS: Rat conjunctival goblet cells and CGCs contain all NTs and NTrs. Only NGF and BDNF stimulated goblet cell glycoconjugate secretion, and none induced CGC proliferation.     

Expression and localisation of BDNF, NT4 and TrkB in proliferative vitreoretinopathy

Exogenous brain derived neurotrophic factor (BDNF) is known to rescue ganglion cell death after optic nerve injury. Its mechanism of action is believed to be indirect via glial cells in the retina. In this study we investigated the changes in expression and localisation of BDNF, neurotrophin-4 (NT4) and their common receptor (TrkB) in retinectomy sections of patients with proliferative vitreoretinopathy (PVR). Nine full-thickness retinectomy specimens obtained at retinal reattachment surgery for PVR were fixed in 4% paraformaldehyde immediately after excision and compared to similarly processed normal donor retinas (4 eyes). Agarose-embedded sections (100 microm thick) were double labelled for immunohistochemistry by confocal microscopy, with antibodies against BDNF, NT4, TrkB, rod opsin, glial fibrillary acidic protein (GFAP), cellular retinaldehyde binding protein (CRALBP) and Brn3. This study demonstrates expression of NT4 by ganglion cells and shows expression of BDNF and NT4 in the outer photoreceptor segments is downregulated during PVR, whilst NT4 is markedly upregulated throughout the retina during this condition. The findings here suggest that NT4 may play a neural protective role during the development of PVR. It also shows that upregulation of NT4 in PVR is localised to Müller glial cells, indicating either over-expression of this factor by Müller cells or that Müller cells internalise NT4 for trafficking across the retina. TrkB expression was not observed in PVR retina. The observations that Müller glia demonstrate upregulation of NT4 suggests that retinal injury may lead to activation of this neurotrophin by Müller cells as part of their neuroprotective functions.     

Role of neurotrophin-4/5 in neural cell death during retinal development and ischemic retinal injury in vivo

PURPOSE: Neurotrophin (NT)-4/5 and brain-derived neurotrophic factor (BDNF) mediate cell survival through TrkB, a high-affinity tyrosine kinase receptor, and may prevent neural cell death in various pathologic conditions. This study was conducted to investigate the function of NT-4/5 in neural cell death during retinal development and ischemic retinal injury. METHODS: Retinal development in wild-type, NT-4/5 knockout (KO), and NT-4/5:BDNF double-KO mice was histologically examined from postnatal day 0 (P0) to P90. Ischemic retinal injury was performed at P42, and NT-4/5 mRNA expression level and the extent of retinal cell death was quantitatively examined. RESULTS: Real-time PCR analysis revealed increased NT-4/5 mRNA expression in the ischemic retina. In the NT-4/5 KO mouse, retinal development and structure were normal, but the strain was susceptible to ischemic injury on P42. In contrast, NT-4/5:BDNF double-KO mice showed delayed retinal development and died before P42. CONCLUSIONS: These results suggest that NT-4/5, in combination with other trophic factors, is involved in the postnatal survival of retinal neurons during both development and degeneration.     

NADPH-diaphorase colocalization with somatostatin receptor subtypes sst2A and sst2B in the retina

PURPOSE: To investigate the differential localization of somatotropin release-inhibitory factor (SRIF) receptor subtypes (sst2A and sst2B) and their possible colocalization with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in the rat and rabbit retina. METHODS: Polyclonal antibodies raised against sst2A and sst2B receptors were applied to 10- to 14-microm cryostat sections of rat and rabbit retinas fixed in paraformaldehyde. NADPH-diaphorase reactivity was assessed histochemically. Double labeling was performed for sst2A or sst2B receptors with NADPH-diaphorase, and with markers for the cell types present in the retina (protein kinase C [PKC], tyrosine hydroxylase; [TH], calbindin, and recoverin). RESULTS: sst2A immunoreactivity was detected in rod bipolar cells and colocalized with NADPH-diaphorase in the rabbit, but not the rat, retina. sst2B was present only in photoreceptor cells of the rat and colocalized with NADPH-diaphorase. CONCLUSIONS: These results suggest that SRIF, acting through sst2A receptors in bipolar cells and sst2B receptors in photoreceptor cells, may affect nitric oxide function in the rabbit and rat retina.     

Rat retinal ganglion cells co-express brain derived neurotrophic factor (BDNF) and its receptor TrkB.

The expression of brain derived neurotrophic factor (BDNF) and its preferred receptor (TrkB) in rat retinal ganglion cells (RGCs) have been determined in the present study. To identify RGCs retrograde labelling was performed with fluorogold (FG). Subsequently, retinas were immunostained with antibodies to BDNF and TrkB. We found that all RGCs labelled with FG express both BDNF and its preferred receptor, TrkB. Moreover, displaced amacrine cells were also found to be immunolabelled by both antibodies. Thus BDNF/TrkB signalling in RGCs probably involves endogenous BDNF produced by the RGCs themselves.     

大鼠视网膜神经网膜上神经营养因子的表达

目的　探讨神经生长因子 (N GF)、神经营养因子 3 (N T- 3 )及脑源性神经营养因子 (BDN F)在大鼠神经视网膜上的表达与分布。方法　 4 0 g· L- 1多聚甲醛灌注成年 Wistar大鼠 ,断头取眼球 ,放入 4 0 g· L- 1多聚甲醛 -蔗糖 (3 0 0 g· L- 1)固定液中过夜。运用免疫组化方法定性测定视网膜神经网膜上 N GF、BDN F、N T- 3的表达与分布。结果　 NG F、BDN F、NT - 3在 W istar大鼠视网膜神经网膜内均有表达 ,而且 3种神经营养因子主要分布于神经节细胞层和内核层 ,NT- 3主要分布于细胞核内 ,而N GF和 BDN F主要分布于细胞浆内。结论　 W istar大鼠视网膜神经网膜内含有丰富的神经营养因子 N GF、BDN F、N T- 3。     

Cone photoreceptors in laminated retinal transplants.

PURPOSE: To investigate the contents of green- and blue-sensitive cone photoreceptors in laminated rabbit retinal transplants. METHODS: Eleven rabbits each received a sheet of embryonic neuroretina into the subretinal space in one eye. Vitrectomy was used in the procedure and properly polarized flat transplants were placed on the host pigment epithelium. After 17-309 days the transplants were examined immunohistochemically with specific antibodies against COS-1 (green-sensitive cones) and OS-2 (blue-sensitive cones). RESULTS: All grafts displayed normal lamination with well developed photoreceptor outer segments apposed to the host retinal pigment epithelium. Occasionally, rosettes were found at the transplant edges. Both COS-1 positive and OS-2 positive cones were detected. In the laminated part of the grafts, COS-1 positive cones were more numerous than OS-2 positive ones. In the rosetted parts of the transplants the relationship between the cones was reversed. CONCLUSION: Full-thickness embryonic rabbit retinal transplants develop into laminated retinas with well-developed photoreceptor outer segment. Both green- and blue-sensitive cone photoreceptors are present and the ratio between the two cone types is the same as in the normal adult rabbit retina.     

Obstructed axonal transport of BDNF and its receptor TrkB in experimental glaucoma

PURPOSE: In both animal model system and in human glaucoma, retinal ganglion cells (RGCs) die by apoptosis. To understand how RGC apoptosis is initiated in these systems, the authors studied RGC neurotrophin transport in experimental glaucoma using acute intraocular pressure (IOP) elevations in rats and chronic IOP elevation and unilateral optic nerve transections in monkeys. METHODS: Eyes were studied in masked fashion by light and electron microscopy and by immunohistochemistry with antibodies directed against the tyrosine kinase receptors (TrkA, B, and C) and against brain-derived neurotrophic factor (BDNF), as well as by autoradiography to identify retrograde axonal transport of 125I-BDNF injected into the superior colliculus. RESULTS: With acute glaucoma in the rat, RGC axons became abnormally dilated, accumulating vesicles presumed to be moving in axonal transport at the optic nerve head. Label for TrkB, but not TrkA, was relatively increased at and behind the optic nerve head with IOP elevation. Abnormal, focal labeling for TrkB and BDNF was identified in axons of monkey optic nerve heads with chronic glaucoma. With acute IOP elevation in rats, radiolabeled BDNF arrived at cells in the RGC layer at less than half the level of control eyes. CONCLUSIONS: Interruption of BDNF retrograde transport and accumulation of TrkB at the optic nerve head in acute and chronic glaucoma models suggest a role for neurotrophin deprivation in the pathogenesis of RGC death in glaucoma.     

Two cone types of rat retina detected by anti-visual pigment antibodies.

The presence of two distinct cone types was demonstrated in the retina of the rat using two cone-specific monoclonal anti-visual pigment antibodies. Cones labelled by antibody COS-1 constituted the large majority (about 93%) of cones, and are most probably responsible for the green photopic sensitivity of the rat. About 7% of the cones were recognized by antibody OS-2, and are thought to be blue-sensitive elements. While OS-2 positive cones were evenly distributed throughout the retina, there were slight differences in the distribution of COS-1 positive cones. The cones made up about 0.85% of all photoreceptor cells. Although the OS-2 positive cones occur in a very low number (0.05% of all photoreceptors) and probably do not appreciably contribute to the photopic system of the rat, their presence in the rat strengthens the presumption that most mammalian species exhibit a dual cone system with a shortwave and a middle-to-longwave sensitivity.     

Localization of iodopsin in the chick retina during in vivo and in vitro cone differentiation

Using highly specific antibodies against a chick red-sensitive cone pigment, iodopsin, we investigated the localization of iodopsin in the developing and mature chick retina. The chick retina contains several different photoreceptor types, including a rod, a double cone with a principal and accessory cone, and four different types of single cones. Immunocytochemical observations revealed that outer segments (OS) of one of the single cones (type 1) and both cells of the double cone were strongly immunoreactive to anti-iodopsin antibodies. The Golgi regions and small vesicular structures in the inner segments (IS) of these cells also were intensely stained, indicating a continuous synthesis of iodopsin and its addition to the newly formed cone OS. In the differentiating cones of the developing but immature chick retina, iodopsin immunoreactivity was found at the plasma membranes of both the IS and the terminals (pedicles). This suggests that unidirectional transport of iodopsin to the outer segment may be established during cone differentiation. Immunostaining in the outer plexiform layer (OPL) produced two bands, suggesting that the pedicles of the double cones and type 1 single cones terminate at different positions in this layer. Application of the antibodies to a cell culture system of the chick retina revealed that cells immunoreactive to anti-iodopsin differ slightly in morphology from those reactive with anti-rhodopsin. Since antibodies to iodopsin and rhodopsin stained different types of photoreceptors in the intact chick retina, it will be possible to analyze cell lineage of rods and cones in vitro by use of these antibodies.     

Immunocytochemical reactivity of rod and cone visual pigments in the sturgeon retina.

Microspectrophotometry and immunocytochemistry with several antivisual pigment antibodies were used to study visual cells of the Siberian sturgeon, Acipenser baeri Brandt. The retina contained rods and three morphological types of cones: large cones with oil drops, small cones with oil drops, and cone-like cells without oil drops. Rods and cone-like drop-free cells were found to possess porphyropsin-549, while the large oil drop-bearing cones contained red-sensitive (P613), green-sensitive (P542), and blue-sensitive (P462) visual pigments. The immunocytochemical staining pattern with three antibodies to visual pigment proteins also revealed one visual pigment in rods and three visual pigments in cones. Rods were labeled with all three antibodies, while the majority of large cones (type I), presumably the red-sensitive ones, were negative with the polyclonal serum AO against bovine opsin. A less-frequently occurring large cone type (type II) was stained by all three antibodies including mAb COS-1 specific to middle-to-long-wave visual pigments in birds and mammals, and is thought to be green-sensitive. An even less-frequent large cone type (type III, probably the blue-sensitive one) did not bind COS-1. The small cones with oil droplets showed immunoreactivities similar to either type II or type III cones. The oil drop-free small photoreceptor exhibited a staining pattern identical with that of rods. These results indicate that the immunocytochemical approach can be used to reveal photoreceptor-specific neural connections in the sturgeon retina.     

Cone differentiation with no photopigment coexpression

PURPOSE: To decide whether the transitory coexpression of cone visual pigments described in the developing rat and gerbil retina is a universal feature of dichromatic mammalian species. METHODS: The rabbit, a species widely used in eye research, was selected for the study and a search made for the presence of cones that bound more than one cone antibody during the first postnatal week. To plot the densities of individual cone types and to colocalize the two visual pigments, immunocytochemistry on retinal wholemounts and consecutive tangential sections, respectively, were used. RESULTS: The sequence in which the visual pigments began to be expressed was the same as that observed in other mammals: first, rhodopsin; second, blue pigment; and last, green pigment. The striking increase in blue cone density numbers observed in the rat, however, did not occur in the rabbit. Instead, some days after the first blue cones appeared, the green cones also started to express their visual pigment, and this cone type soon outnumbered the blue cones. Within the limits of the immunocytochemical method, it was established that unlike the developing rat, the presence of double-labeled cones was not a character of the rabbit retina. CONCLUSIONS: Visual pigment coexpression is an interesting phenomenon of retinal development, however, it is not the exclusive scenario of photoreceptor differentiation. Each species must be carefully studied before deciding whether its retinal cones synthesize both pigments during retinal development.     

Temporal and spatial differences in expression of TrkB isoforms in rat retina during constant light exposure

Brain-derived neurotrophic factor (BDNF) has been reported to rescue neuroretinal cells under different toxic conditions. These cells include not only those expressing BDNF receptors (TrkB) but also those not expressing TrkB including photoreceptors. The purpose of this study was to determine the retinal sites at which BDNF and TrkB isoforms are expressed after different durations of continuous light exposure, and to compare these sites with those of TUNEL-positive cells in the same retina. Sprague-Dawley rats were exposed to continuous light for different durations. The expressions of BDNF and TrkB isoforms, TrkB-FL and TrkB-T1, were determined by Western blot analysis, real-time PCR, immunohistochemistry, and in situ hybridization before and after the light exposure. The number of TUNEL-positive cells reached a maximum at 48 to 72h after light exposure. The degree of up-regulation of the TrkB-T1 gene was significantly higher than that in normal control eyes at 24h by real-time PCR. Immunohistochemistry showed that TrkB-FL-positive cells were detected in all retinal layers except the outer nuclear layer (ONL), photoreceptor cells, and retinal pigment epithelium (RPE). The number of TrkB-FL-positive cells in the IPL was transiently decreased at 6h, and was increased on the processes of the Mueller cells in the ONL after 48h. TrkB-T1 was expressed in the INL, OPL, and RPE, and was up-regulated on the soma of Mueller cells after 24h. In situ hybridization showed that the expression of the TrkB-FL gene was up-regulated in the INL after 48h when the number of TUNEL-positive cells was at its peak. The TrkB-T1 gene was up-regulated before or just prior to the appearance of TUNEL-positive cells. These results suggest that BDNF transduces the signals using appropriate receptor isoforms that are expressed temporally and spatially differentially on Mueller cells during light-induced retinal degeneration.     

Green-sensitive cone photoreceptors are selectively labeled by Procion yellow dye in goldfish retina

Selective labeling of intravitreal Procion yellow dye by presumed blue-sensitive cone photoreceptors has been demonstrated in primate retina. To determine whether Procion yellow is selective for this cone type in an unrelated vertebrate species, labeling by this dye was studied in goldfish retina, where cone pigment type can be directly inferred from photoreceptor morphology. At low vitreal concentrations of the dye (less than 0.4%), only cone outer segments were labeled. At vitreal concentrations of 0.4-0.5%, the inner segments of short-double cones and a subset of long single cones (presumed green-sensitive cones) were selectively stained. At still higher vitreal concentrations (0.6-0.7%), the inner segments of short-single cones and miniature short-single cones (presumed blue-sensitive cones) showed evidence of Procion label, but were not as heavily labeled. The inner segments of long-double cones and a subset of long-single cones (presumed red-sensitive cones) did not label at any of these concentrations. These results show that Procion yellow is not a selective marker for blue-sensitive cones in the goldfish retina. In addition, stained rod and cone nuclei were observed at each dye concentration, including those concentrations at which no inner segments were labeled.