Effect of Purified Murine NGF on Isolated Photoreceptors of a Rodent Developing Retinitis Pigmentosa

A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies.     

An extracellular retinol-binding glycoprotein in the eyes of mutant rats with retinal dystrophy: development, localization, and biosynthesis

Interstitial retinol-binding protein (IRBP) is a soluble glycoprotein in the interphotoreceptor matrix of bovine, human, monkey, and rat eyes. It may transport retinol between the retinal pigment epithelium and the neural retina. In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22). In the RCS-rdy+ rats, the amount increased slightly after P23. However, in the RCS rats there was a rapid fall in the quantity of IRBP as the photoreceptors degenerated between P23 and P29. No IRBP was detected by immunocytochemistry in rats at P28. The amount of rhodopsin fell more slowly. Although retinas from young RCS and RCS-rdy+ rats were able to synthesize and secrete IRBP, this ability was lost in retinas from older RCS rats (P51, P88) but not their congenic controls. The photoreceptor cells have degenerated at these ages in the RCS animals, and may therefore be the retinal cells responsible for IRBP synthesis. The putative function of IRBP in the extracellular transport of retinoids during the visual cycle is consistent with a defect in retinol transport in the RCS rat reported by others.     

Bevacizumab Eye Drop Treatment Stimulates Tear Secretion in Rats Through Changes in VEGF and NGF Lacrimal Gland Levels

VEGF and NGF are known to modulate corneal healing, neovascularisation and tear secretion. While a VEGF-NGF cross talk has been recently shown to modulate corneal healing in rats, it is not known whether it also plays a role in the regulation of lacrimal function. In this study we aim to investigate the effects of anti-VEGF eye drop treatment on lacrimal gland function and on the local expression of VEGF and NGF in rats. Tear function was measured in 3 months old rats by modified Schirmer test at baseline and after 3 weeks of topical anti-VEGF eye drop treatment. Whole lacrimal glands from rats were removed after treatment and analysed by ELISA for VEGF and NGF levels. To investigate if the effects of anti-VEGF were mediated by changes in the NGF-pathway, we repeated the experiments in RCS rats, a strain with NGF-pathway impairment associated with decreased tear flow. After topical treatment with anti-VEGF eye drops, an increase in tear secretion was observed in both wild-type and RCS rats. A significant decrease of VEGF levels was also observed in lacrimal glands of both RCS and SD rats, accompanied by a significant increase in NGF levels. Inhibition of VEGF at the ocular surface in rats results in changes of tear function and lacrimal gland levels of VEGF and NGF. Further studies on the VEGF/NGF cross-talk at the ocular surface may expand our knowledge on the pathogenesis of several diseases characterized by tear dysfunction.     

Parvalbumin immunoreactivity is enhanced by brain-derived neurotrophic factor in organotypic cultures of rat retina.

The rodent retina undergoes considerable postnatal neurogenesis and phenotypic differentiation, and it is likely that diffusible neurotrophic factors contribute to this development and to the subsequent formation of functional retinal circuitry. Accordingly, perturbation of specific neurotrophin ligand-receptor interactions has provided valuable information as to the fundamental processes underlying this development. In the present studies we have built upon our previous observation that suppression of expression of trk(B), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the postnatal rat retina results in the alteration of a specific interneuron in the rod pathway-the parvalbumin (PV)-immunoreactive AII amacrine cell. Here, we isolated retinas from newborn rats and maintained them in organotypic culture for up to 14 days (approximating the time of eye opening, in vivo) in the presence of individual neurotrophins [BDNF or nerve growth factor (NGF)]. We then examined histological sections of cultures for PV immunoreactivity. In control cultures, only sparse PV-immunostained cells were observed. In cultures supplemented with NGF, numerous lightly immunostained somata were present in the inner nuclear layer (INL) at the border of the inner plexiform layer (IPL). Many of these cells had rudimentary dendritic arborizations in the IPL. Cultures supplemented with BDNF displayed numerous well-immunostained somata at the INL/IPL border that gave rise to elaborate dendritic arborizations that approximated the morphology of mature AII amacrine cells in vivo. These observations indicate that neurotrophins have specific effects upon the neurochemical and, perhaps, morphological differentiation of an important interneuron in a specific functional retinal circuit.     

Age-dependence of the nerve fibre growth-promoting effects of hippocampus and exogenous nerve growth factor on cultured rat septum and superior cervical ganglion

Explants of hippocampus from rats at various ages evoked an intense nerve fibre growth from cocultured superior cervical ganglion and septum explants taken from newborn rats. The addition of antiserum to nerve growth factor (NGF) into the culture medium inhibited the outgrowth of nerve fibres from superior cervical ganglia, while septum explants still extended nerve fibres in the same medium.Septum explants responded to added NGF, as well as to cocultured hippocampus, during the first postnatal week only, whereas ganglia extended nerve fibres in NGF-containing cultures throughout the postnatal period and even at the age of 6 months if superoptimal concentration of NGF was used.The present results suggest that hippocampus releases NGF and some other growth factor(s) in culture throughout the postnatal period from birth to adulthood. On the other hand, the capacity of septum to extend nerve fibres in response to the growth factors appears to be restricted to the first postnatal week.     

Congenic strains of RCS rats with inherited retinal dystrophy.

Two congenic strains of RCS rats, RCS-p/+ and RCS-c, have been developed that differ from the parental strain at genetic loci affecting pigmentation. Inbred RCS rats are pink-eyed, while RCS-p/+ rats produce segregating litters of pink-eyed (p/p) and black-eyed (p/+) offspring, and RCS-c rats are albinos. All the strains are homozygous for the mutant form of the retinal dystrophy gene. The black eye pigment in RCS-p/+ rats slows the progression of the retinal degeneration by about 10 days in the posterior retina and by about 30-35 days in the peripheral retina in the superior half of the eye. No slowing of the disease occurs in the inferior half of the eye along the vertical meridian. All the strains are similar in body weight and litter size, and show a low incidence of cataract and microphthalmia.     

Microglia-derived pronerve growth factor promotes photoreceptor cell death via p75 neurotrophin receptor

Reports implicating microglia-derived nerve growth factor (NGF) during programmed cell death in the developing chick retina led us to investigate its possible role in degenerative retinal disease. Freshly isolated activated retinal microglia expressed high molecular weight forms of neurotrophins including that of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. Conditioned media from cultured retinal microglia (MGCM) consistently yielded a approximately 32-kDa NGF-reactive band when supplemented with bovine serum albumin (BSA) or protease inhibitors (PI); and promoted cell death that was suppressed by NGF immunodepletion in a mouse photoreceptor cell line (661w). The approximately 32 kDa protein was partially purified (MGCM/p32) and was highly immunoreactive with a polyclonal anti-pro-NGF antibody. Both MGCM/p32 and recombinant pro-NGF protein promoted cell death in 661w cultures. Increased levels of pro-NGF mRNA and protein were observed in the RCS rat model of retinal dystrophy. MGCM-mediated cell death was reversed by p75NTR antiserum in p75NTR(+)/trkA(-) 661w cells. Our study shows that a approximately 32 kDa pro-NGF protein released by activated retinal microglia promoted degeneration of cultured photoreceptor cells. Moreover, our study suggests that defective post-translational processing of NGF might be involved in photoreceptor cell loss in retinal dystrophy.     

Parvalbumin immunoreactivity is enhanced by brain‐derived neurotrophic factor in organotypic cultures of rat retina

The rodent retina undergoes considerable postnatal neurogenesis and phenotypic differentiation, and it is likely that diffusible neurotrophic factors contribute to this development and to the subsequent formation of functional retinal circuitry. Accordingly, perturbation of specific neurotrophin ligand–receptor interactions has provided valuable information as to the fundamental processes underlying this development. In the present studies we have built upon our previous observation that suppression of expression of trk_B, the high‐affinity receptor for brain‐derived neurotrophic factor (BDNF), in the postnatal rat retina results in the alteration of a specific interneuron in the rod pathway—the parvalbumin (PV)‐immunoreactive AII amacrine cell. Here, we isolated retinas from newborn rats and maintained them in organotypic culture for up to 14 days (approximating the time of eye opening, in vivo) in the presence of individual neurotrophins [BDNF or nerve growth factor (NGF)]. We then examined histological sections of cultures for PV immunoreactivity. In control cultures, only sparse PV‐immunostained cells were observed. In cultures supplemented with NGF, numerous lightly immunostained somata were present in the inner nuclear layer (INL) at the border of the inner plexiform layer (IPL). Many of these cells had rudimentary dendritic arborizations in the IPL. Cultures supplemented with BDNF displayed numerous well‐immunostained somata at the INL/IPL border that gave rise to elaborate dendritic arborizations that approximated the morphology of mature AII amacrine cells in vivo. These observations indicate that neurotrophins have specific effects upon the neurochemical and, perhaps, morphological differentiation of an important interneuron in a specific functional retinal circuit. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 376–384, 1999     

Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa

Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP.     

Asthma pregnancy alters postnatal development of chromaffin cells in the rat adrenal medulla

Background

Adrenal neuroendocrine plays an important role in asthma. The activity of the sympathoadrenal system could be altered by early life events. The effects of maternal asthma during pregnancy on the adrenal medulla of offspring remain unknown.

Methodology/Principal Findings

This study aims to explore the influence of maternal asthma during pregnancy on the development and function of adrenal medulla in offspring from postnatal day 3 (P3) to postnatal day 60 (P60). Asthmatic pregnant rats (AP), nerve growth factor (NGF)-treated pregnant rats (NP) and NGF antibody-treated pregnant rats (ANP) were sensitized and challenged with ovalbumin (OVA); NP and ANP were treated with NGF and NGF antibody respectively. Offspring rats from the maternal group were divided into four groups: offspring from control pregnant rats (OCP), offspring from AP (OAP), offspring from NP (ONP), and offspring from ANP (OANP). The expressions of phenylethanolamine N-methyltransferase (PNMT) protein in adrenal medulla were analyzed. The concentrations of epinephrine (EPI), corticosterone and NGF in serum were measured. Adrenal medulla chromaffin cells (AMCC) were prone to differentiate into sympathetic nerve cells in OAP and ONP. Both EPI and PNMT were decreased in OAP from P3 to P14, and then reached normal level gradually from P30 to P60, which were lower from birth to adulthood in ONP. Corticosterone concentration increased significantly in OAP and ONP.

Conclusion/Significance

Asthma pregnancy may promote AMCC to differentiate into sympathetic neurons in offspring rats and inhibit the synthesis of EPI, resulting in dysfunction of bronchial relaxation.     

Changes in the properties of photostimulated cyclic nucleotide phosphodiesterase from the retina of rats with hereditary retinal degeneration

A degree of extractability and activation of cGMP-phosphodiesterase (PDE) (EC. 3.1.4.17) from the rod outer segment membranes was studied in Campbell rats with inherited retinal degeneration and control Wistar rats as compared to the control, the PDE extractability in the diseased rats was found to be considerably lower, which manifested as early as the 15th day of the postnatal life. Changes in the GTP-stimulated and basal PDE activity were observed in Campbell rats. Beginning from the 25th day of the postnatal life the GTP-stimulated PDE of degenerative retina decreased and by the 60th day it reached the basal activity level in these animals. In the diseased rats the first 57 days of postnatal life the basal activity of PDE was sufficiently higher, followed by a sharp decrease reaching the basal activity level of the control rats. The obtained data on the changed PDE activity are likely to be a result of the disturbance in the protein-lipid interaction and a change in the external layer of the photoreceptor membranes in rats with inherited retinal degeneration.     

Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration

Purpose

To investigate the safety and efficacy of subretinal injection of human Wharton’s Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats.

Methods

RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8) and placebo control group (n = 8). In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT). Retinal function was assessed by electroretinography (ERG) 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies.

Results

No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL) in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells.

Conclusions

Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies.     

Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats

Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats.S.-P.H. and P.-K.L. contributed equally to this work.     

Presence and possible functional role of nerve growth factor in the thymus.

We have previously reported that the nerve growth factor (NGF), a polypeptide known for its neurotrophic activities, is also involved in proliferation, growth and survival of cells of the immune system. Working with animal models, we found that NGF and NGF-receptors (NGF-r) are present in the cells of the medullary layer of the thymus, a lymphoid gland involved in the production and differentiation of T-lymphocytes. Using immunohistochemical and biochemical approaches, we also showed that the expression of NGF in the thymus is high during late prenatal life and decreases later in postnatal life. A significant alteration of NGF levels was also found during pregnancy and aging, two events characterized by thymic involution. The aim of this study is to investigate whether NGF and NGF-r expression in the thymus are influenced by immuno- and neuro-pathological events. These observations will be presented and discussed.     

Ocular distribution of 70-kDa heat-shock protein in rats with normal and dystrophic retinas

Summary Stress proteins are thought to play an important role in cellular development and in survival mechanisms. We compared the immunolocalization of the 70-kDa stress protein (SP70) in the ocular tissue of the normal Sprague-Dawley (SD) rat with that in the Royal College of Surgeons (RCS) rat with retinal dystrophy. SP70 was present in the maturing ocular tissues of both rat strains. However, once retinal degeneration began in the RCS rat, the retinal pigment epithelium and photoreceptor cells showed increased immunostaining for SP70 over that observed in age-matched SD rats. In late stages of retinal degeneration, immunostaining for SP70 was considerably reduced in the RCS retina, whereas normal distribution of immunostaining for SP70 in the SD retina was preserved, albeit decreased, through postnatal day 180. The optic nerve, ciliary body, and corneal epithelium were also influenced by the dystrophic disease condition, although the pattern of changes in SP70 immunostaining differed for each tissue. These results suggest that the genetic defect in the RCS rat produces a state of metabolic stress in all ocular tissues as the degeneration progresses, but that the subsequent rise in ocular SP70 is insufficient to prevent progression of the disease.     

Early postnatal exposure of mice to side-steam tobacco smoke increases neuropeptide Y in lung

Our recent study showed that prenatal and early postnatal exposure of mice to side-steam tobacco smoke (SS), a surrogate to environmental tobacco smoke (ETS), leads to increased airway responsiveness and sensory innervation later in life. However, the underlying mechanism initiated in early life that affects airway responses later in life remains undefined. The concomitant increase in nerve growth factor (NGF) after exposures suggests that NGF may be involved the regulation of airway innervation. Since NGF regulates sympathetic nerve responses, as well as sensory nerves, we extended previous studies by examining neuropeptide Y (NPY), a neuropeptide associated with sympathetic nerves. Different age groups of mice, postnatal day (PD) 2 and PD21, were exposed to either SS or filtered air (FA) for 10 consecutive days. The level of NPY protein in lung and the density of NPY nerve fibers in tracheal smooth muscle were significantly increased in the PD2-11SS exposure group compared with PD2-11FA exposure. At the same time, the level of NGF in lung tissue was significantly elevated in the PD2-11SS exposure groups. However, neither NPY (protein or nerves) nor NGF levels were significantly altered in PD21-30SS exposure group compared with the PD21-30FA exposure group. Furthermore, pretreatment with NGF antibody or K252a, which inhibits a key enzyme (tyrosine kinase) in the transduction pathway for NGF receptor binding, significantly diminished SS-enhanced NPY tracheal smooth muscle innervation and the increase in methacholine-induced airway resistance. These findings show that SS exposure in early life increases NPY tracheal innervation and alters pulmonary function and that these changes are mediated through the NGF.     

Developmental regulation of nerve growth factor and its receptor in the rat caudate-putamen

In prior studies, nerve growth factor (NGF) administration induced a robust, selective increase in the neurochemical differentiation of caudate-putamen cholinergic neurons. In this study, expression of NGF and its receptor was examined to determine whether endogenous NGF might serve as a neurotrophic factor for these neurons. The temporal pattern of NGF gene expression and the levels of NGF mRNA and protein were distinct from those found in other brain regions. NGF and high-affinity NGF binding were present during cholinergic neurochemical differentiation and persisted into adult-hood. An increase in NGF binding during the third postnatal week was correlated with increasing choline acetyltransferase activity. The data are consistent with a role for endogenous NGF in the development and, possibly, the maintenance of caudate-putamen cholinergic neurons.     

Nerve growth factor preserves a critical motor period in rat striatum

We previously found the occurrence of a critical motor period during rat postnatal development where circling training starting the 7-day schedule at 30 days-but not before or after-induces a lifetime drop in the binding to cholinergic muscarinic receptors (mAChRs) in striatum. Here, we studied whether nerve growth factor (NGF) participates in this restricted period of muscarinic sensitivity. For this purpose, we administered mouse salival gland 2.5S NGF (1.4 or 0.4 microg/day, infused by means of ALZA minipumps) by intrastriatal unilateral route between days 25 and 39, and then trained rats starting at 40 days. Under these conditions, NGF induced a long-term reduction in the striatal [3H] quinuclidilbenzylate (QNB) binding sites despite the fact that motor training was carried out beyond the natural critical period. Thus, at day 70, measurement of specific QNB binding in infused striata of trained rats showed decreases of 42% (p < .0004) and 33% (p < .02) after administration of the higher and lower NGF doses, respectively, with respect to trained rats treated with cytochrome C, for control. Noncannulated striata of the NGF-treated rats also showed a decrease in QNB binding sites (44%; p < .0001) only at the higher infusion rate. This effect was not found in the respective control groups. Our observations show that NGF modulates the critical period in which activity-dependent mAChR setting takes place during rat striatal maturation.     

Inhibitory Peptide of Mitochondrial μ-Calpain Protects against Photoreceptor Degeneration in Rhodopsin Transgenic S334ter and P23H Rats

Mitochondrial μ-calpain and apoptosis-inducing factor (AIF)-dependent photoreceptor cell death has been seen in several rat and mouse models of retinitis pigmentosa (RP). Previously, we demonstrated that the specific peptide inhibitor of mitochondrial μ-calpain, Tat-µCL, protected against retinal degeneration following intravitreal injection or topical eye-drop application in Mertk gene-mutated Royal College of Surgeons rats, one of the animal models of RP. Because of the high rate of rhodopsin mutations in RP patients, the present study was performed to confirm the protective effects of Tat-µCL against retinal degeneration in rhodopsin transgenic S334ter and P23H rats. We examined the effects of intravitreal injection or topical application of the peptide on retinal degeneration in S334ter and P23H rats by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electroretinogram (ERG), immunohistochemistry for AIF, and histological staining. In S334ter rats, we found that intravitreal injection or topical application of the peptide prevented photoreceptor cell death from postnatal (PN) 15 to 18 days, the time of early-stage retinal degeneration. Topical application of the peptide also delayed attenuation of ERG responses from PN 28 to 56 days. In P23H rats, topical application of the peptide protected against photoreceptor cell death and nuclear translocation of AIF on PN 30, 40, and 50 days, as the primary stages of degeneration. We observed that topical application of the peptide inhibited the thinning of the outer nuclear layer and delayed ERG attenuations from PN 30 to 90 days. Our results demonstrate that the mitochondrial μ-calpain and AIF pathway is involved in early-stage retinal degeneration in rhodopsin transgenic S334ter and P23H rats, and inhibition of this pathway shows curative potential for rhodopsin mutation-caused RP.     

Developmental changes in the responses of rat chromaffin cells to neuronotrophic and neurite-promoting factors

This study describes the survival and neurite outgrowth behaviors of cultured adrenal medullary (chromaffin) cells obtained from postnatal rats 1 day (D1) to 100 days (D100) old in response to nerve growth factor (NGF), chick eye ciliary neuronotrophic factor (CNTF), and laminin. In the absence of trophic factors the 4-day survival of cultured chromaffin cells (relative to the number of cells attached at 2 hr) increased from one-third of the cells at D1 to 40% at D8 and 90-100% at D16 and older stages. At saturating concentrations NGF increased cell survival at D8 by 90%, but failed to support all chromaffin cells present at 2 hr. In contrast, CNTF supported the survival of all cells at D8. At D1 NGF and CNTF had only a very small effect on survival during the 4-day culture period, although both factors clearly enhanced the numbers of surviving cells after 8 days. Either NGF or CNTF also elicited neurite outgrowth from rat chromaffin cells, which amounted to approximately 15-20% at D1 and D8 and subsequently decreased to about 5-8% at D30 and virtually zero at D100. At this last age both factors applied together clearly elicited neurites. Such a potentiating effect of NGF and CNTF was also seen at earlier postnatal ages. Laminin did not affect neurite growth at D30 in the absence of trophic factors, as already described for D8 rat chromaffin cells. In the presence of NGF, however, it increased neurite length and branching during a 4-day culture period and even enhanced neurite recruitment at later culture times. These data suggest that rat chromaffin cells undergo age-related changes in their responses to NGF and CNTF and that laminin modulates their neurite outgrowth behaviors in the presence of trophic factors.