Targeted surface marker screening on neuronal structures in the human choroid.

While choroidal neuronal control is known to be essential for retinal and ocular health, its mechanisms are not understood. Especially, the local choroidal innervation mediated by intrinsic choroidal neurons (ICN) remains enigmatic. Neuronal functionality depends on the synaptic neurotransmitters and neuroregulatory peptides involved as well as from membrane components presented on the cell surface. Since the neuronal surface molecular expression patterns in the choroid are currently unknown, we sought to determine the presence of various cluster-of-differentiation (CD) antigens in choroidal neuronal structures with a particular focus on ICN. Human choroids were prepared for immunohistochemistry and the pan-neuronal marker PGP9.5 was combined with CD15, CD24, CD29, CD34, CD46, CD49b, CD49e, CD56, CD58, CD59, CD71, CD81, CD90, CD146, CD147, CD151, CD165, CD171, CD184, CD200, CD271 and fluorescence- and confocal laser scanning-microscopy was used for documentation. The following antigens were found to be co-localized in PGP.9.5+ nerve fibers and ICN perikarya: CD29, CD34, CD56, CD81, CD90, CD146, CD147, CD151, CD171, CD200 and CD271, while all other CD markers where not detectable. Whereas CD24- and CD59- immunoreactivity was clearly absent in ICN perikarya, some neural processes of the choroidal stroma displayed CD24 and CD59 immunopositivity. While a multitude of the aforementioned CD-markers were indeed detected in nervous structures of the choroid, the CD24+ and CD59+ nerve fibers most likely have extrinsic origin from cranial ganglia since ICN cell bodies were found to lack both markers. These findings illustrate how the detailed analysis of CD molecules described here opens novel avenues for future functional studies on choroidal innervation and its control.

Effects of autonomic denervations on the rhythms in axial length and choroidal thickness in chicks.

In chicks, axial length and choroidal thickness undergo circadian oscillations. The choroid is innervated by both branches of the autonomic nervous system, but their contribution(s) to these rhythms is unknown. We used two combination lesions to test this. For parasympathectomy, nerve VII was sectioned presynaptic to the pterygopalatine ganglia, and the ciliary post-ganglionics were cut (double lesion; n = 8). Triple lesions excised the sympathetic superior cervical ganglion as well (n = 8). Sham surgery was done in controls (n = 7). 8-14 days later, axial dimensions were measured with ultrasonography at 4-h intervals over 24 h. Rhythm parameters were assessed using a "best fit" function, and growth rates measured. Both types of lesions resulted in ultradian (> 1 cycle/24 h) rhythms in choroidal thickness and axial length, and increased vitreous chamber growth (Exp-fellow: double: 69 µm; triple: 104 µm; p < 0.05). For double lesions, the frequency was 1.5 cycles/day for both rhythms; for triples the choroidal rhythm was 1.5 cycles/day, and the axial was 3 cycles/day. For double lesions, the amplitudes of both rhythms were larger than those of sham surgery controls (axial: 107 vs 54 µm; choroid: 124 vs 29 µm, p < 0.05). These findings provide evidence for the involvement of abnormal ocular rhythms in the growth stimulation underlying myopia development.

VIP changes during daytime in chicken intrinsic choroidal neurons.

PURPOSE: Ocular autonomic control is mediated by sympathetic and parasympathetic nerve fibres. Their interactions are complemented by primary afferent nerve fibers of and intrinsic choroidal neurons (ICN). As the vasodilatative neuropeptide, vasoactive intestinal peptide (VIP), is expressed in extrinsic and intrinsic ocular neurons, it is of special interest in ophthalmic research. Since circadian changes of ocular blood flow are known in humans and birds, this study aimed at investigating VIP expression at different daytimes in chicken choroid, the preferred model species in ICN research. METHODS: 12 eyes of 12 chickens were retrieved, slaughtered at 8.00-9.30 a.m. (n = 6) and 8.00 p.m. (n = 6), respectively, and choroidal wholemounts were prepared for immunofluorescence of VIP. VIP-positive ICN of both groups were quantified and density of VIP-positive axons assessed semi-quantitatively. In 28 additional eyes retrieved in the morning (n = 14) and evening (n = 14), choroidal VIP content was determined by ELISA. Morning and evening data were analyzed statistically. NADPH-diaphorase (NADPH-d, ICN cell marker) was done at additional 12 whole mount choroids of 12 chicken, retrieved in the morning (n = 6) and evening (n = 6). RESULTS: (1) Numbers of VIP positive neurons differed significantly between morning: (239.17 ±â€¯113.9) and evening: (550.83 ±â€¯245.7; p = 0.018). (2) Numbers of VIP-positive perikarya were significantly more accumulated in the temporal part of the choroid in the evening than in the morning (p = 0.026). (3) VIP positive axon density was found to be similar throughout the choroid in the morning and evening. (4) Number of NADPH-d positive neurons was not significantly different between morning (848.8 ±â€¯399.5) and evening (945.8 ±â€¯622.1, p > 0.05). (5) ELISA demonstrated a significant difference of VIP content (p = 0.012) in tissues harvested in the morning (145.41 ±â€¯43.3 pg/ml) compared to evening (221.44 ±â€¯106.3 pg/ml). CONCLUSIONS: As VIP positive axon density was similar in the morning and the evening throughout the choroid, PPG and ICN seemed to contribute equally to the axon network. Yet, changes in the total choroidal VIP content, the numbers of VIP positive perikarya, reflecting the intracellular VIP content, and their topographical distribution at two different days-times argue for a different status of activation of both neuronal sources in contrast to the equal amount of NADPHD-d positive neurons. The higher VIP content in the evening, compared to the morning, correlates with a known circadian rhythm of a lower IOP and a higher choroidal thickness at night. Thus, these changes may argue for a potential role of ICN in the regulation of ocular homeostasis and integrity.

Markers of the sympathetic, parasympathetic and sensory nervous system are altered in the human diabetic choroid.

BACKGROUND: We sought to evaluate alterations in markers of the autonomic nervous system in human diabetic choroid. METHODS: Eighteen eyeballs from subjects with diabetes and 22 eyeballs from subjects without diabetes were evaluated in this study. Synaptophysin, tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DßH), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), vesicular monoamine transporter II (VMAT-2), vesicular acetylcholine transporter (VAChT), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) levels were detected by western blot analysis and immunofluorescence was performed in some cases. Furthermore, differences in adrenergic (α1- and ß2-subtypes) and cholinergic (M1 and M3) receptor levels between diabetic subjects and controls were noted. RESULTS: Decreased synaptophysin levels were found in diabetic choroids by western blot analysis and a reduction of synaptophysin-immunoreactive nerves was also found by immunofluorescence. Furthermore, a decrease of the levels of the key enzyme (TH) and transporter (VMAT2) of norepinephrine was evident both by western blot analysis and immunofluorescence. Additionally, increased NPY, VAChT, nNOS, and CGRP levels were observed in diabetic choroids. The levels of adrenergic (ß2 subtype) and acetylcholine (M1 subtype) receptors decreased in diabetic choroids, as shown by western blotting and although the differences in α1 and M3 were not significant, there was a downward trend. CONCLUSIONS: In the diabetic choroid, the levels of neurotransmitters, enzymes, and receptors associated with choroidal blood flow regulation are altered. These changes may affect the regulation of choroidal blood flow and may be associated with impaired retinal function and retinal pathology.

Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input.

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-beta-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and alpha-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.

Receptors with opposing functions are in postsynaptic microdomains under one presynaptic terminal.

Fast excitatory synaptic transmission through vertebrate autonomic ganglia is mediated by postsynaptic nicotinic acetylcholine receptors (nAChRs). We demonstrate a unique postsynaptic receptor microheterogeneity on chick parasympathetic ciliary ganglion neurons-under one presynaptic terminal, nAChRs and glycine receptors formed separate but proximal clusters. Terminals were loaded with [3H]glycine via the glycine transporter-1 (GlyT-1), which localized to the cholinergic presynaptic terminal membrane; depolarization evoked [3H]glycine release that was calcium independent and blocked by the GlyT-1 inhibitor sarcosine. Ganglionic synaptic transmission mediated by nAChRs was attenuated by glycine. Coexistence of separate clusters of receptors with opposing functions under one terminal contradicts Dale's principle and provides a new mechanism for modulating synaptic activity in vivo.

Defective Choroidal Blood Flow Baroregulation and Retinal Dysfunction and Pathology Following Sympathetic Denervation of Choroid.

Purpose: We sought to determine if sympathetic denervation of choroid impairs choroidal blood flow (ChBF) regulation and harms retina. Methods: Rats received bilateral superior cervical ganglionectomy (SCGx), which depleted choroid of sympathetic but not parasympathetic innervation. The flash-evoked scotopic ERG and visual acuity were measured 2 to 3 months after SCGx, and vasoconstrictive ChBF baroregulation during high systemic arterial blood pressure (ABP) induced by LNAME was assessed by laser Doppler flowmetry (LDF). Eyes were harvested for histologic evaluation. Results: ChBF increased in parallel with ABP in SCGx rats over an ABP range of 90% to 140% of baseline ABP, while in sham rats ChBF remained stable and uncorrelated with ABP. ERG a- and b-wave latencies and amplitudes, and visual acuity were significantly reduced after SCGx. In SCGx retina, Müller cell GFAP immunolabeling was upregulated 2.5-fold, and Iba1+ microglia were increased 3-fold. Dopaminergic amacrine cell fibers in inner plexiform layer were reduced in SCGx rats, and photoreceptors were slightly depleted. Functional deficits and pathology were correlated with impairments in sympathetic regulation of ChBF. Conclusions: These studies indicate that sympathetic denervation of choroid impairs ChBF baroregulation during elevated ABP, leading to choroidal overperfusion. This defect in ChBF regulation is associated with impaired retinal function and retinal pathology. As sympathetic ChBF baroregulatory defects have been observed in young individuals with complement factor H (CFH) polymorphisms associated with risk for AMD, our results suggest these defects may harm retina, perhaps contributing to AMD pathogenesis.

Age-related decline in VIP-positive parasympathetic nerve fibers in the human submacular choroid.

PURPOSE: An age-related decline in macular choroidal blood flow (ChBF) occurs in humans. Vasodilatory nerve fibers containing vasoactive intestinal polypeptide (VIP) innervate choroidal blood vessels. The current study was conducted to examine the possibility that an age-related loss of these fibers might occur in the submacular choroid in humans, and thus contribute to a decline in ChBF. METHODS: Macular choroid punches were collected from 35 healthy human donors ranging from 21 to 93 years of age. Choroidal samples were immunolabeled using anti-VIP and the peroxidase-antiperoxidase METHOD: VIP-positive nerve fiber abundance was quantified in up to 12 fields per punch. Fifty macular punches were analyzed, and results for eye pairs were averaged. Choroidal vessel diameter (ChVD) was measured for these same fields. The relationship between age and vessel diameter or VIP-positive fiber abundance was analyzed. Multivariate statistical models were generated correcting for gender, variables related to the tissue specimens, and potential procedural sources of variability. RESULTS: The fully adjusted multivariate models showed a significant age-related reduction in both the VIP-positive fiber abundance (P = 0.0003, adjusted R(2) = 0.51) and ChVD (P < 0.0001, adjusted R(2) = 0.63), with slopes of -0.45 and -0.19, respectively. Adjusting for the same variables, VIP-positive fiber abundance showed a significant direct correlation with ChVD. CONCLUSIONS: The results indicate a significant age-related decline in VIP-positive nerve fibers and vessel diameter in the submacular choroid in disease-free human donor eyes. These findings suggest that a decline in the neural control of ChBF and vessel diameter may explain the reductions in ChBF and its adaptive control observed clinically with aging.

[A comparative study of choroidal innervation in the human and the rabbit (oryctolagus cuniculus)]. / Estudio comparativo de la inervación coroidea en el hombre y en el conejo (oryctolagus cuniculus).

OBJECTIVE: To analyze morphological differences between the choroidal innervation of the human and the rabbit, the latter being a species frequently used as an experimental model of human ocular diseases. METHODS: Twelve human and 12 rabbit choroidal whole mounts were processed using an indirect immunohistochemical technique, peroxidase-anti-peroxidase and antibodies against 200 kD neurofilament. RESULTS: Choroidal nerve fibers were perivascular and intervascular. Perivascular fibers surrounded all arteries forming a network that was more developed in the rabbit. In humans, intervascular fibers were mainly concentrated at the posterior pole where they formed a denser and more highly organized plexus than in the rabbit, which did not exhibit a preferential location for these fibers. Human choroidal ganglion cells were far more numerous than in the rabbit and were concentrated in a circumferential area corresponding to the entrance of the short posterior ciliary arteries of the submacular area. In the rabbit, these cells were restricted to the peripheral choroid. CONCLUSIONS: Some differences were observed between human and rabbit choroidal innervation. The abundance of ganglion cells and their preferential distribution could be necessary to maintain a constant blood flow in the central area of the human choroid. The lack of organization of rabbit choroidal innervation at the posterior pole could be associated with an absence of the macula. These differences, along with peculiarities of retinal vascularization, should be taken into consideration when using the rabbit as an experimental model to study human eye diseases in which regulation of choroidal blood flow is involved.

NPY and TH innervation in human choroidal whole-mounts.

To determine the distribution of NPY and TH human choroidal innervation, choroidal whole-mounts were processed for indirect immunofluorescence. An antibody to a component of the neuronal cytoskeleton, neurofilament 200 kDa (NF-200) was used to identify neurons and axons. A double immunostaining was performed, antibodies against NF-200 being combined with antibodies against neuropeptide Y (NPY) and tyroxine hydroxylase (TH). Fibers containing both NPY and TH were distributed in three plexuses, one in the suprachoroid large-sized vessel layer, and two in the medium-sized vessel layer. Intrinsic choroidal neurons (ICNs) containing NPY and TH were observed in the suprachoroid. The TH(+) ICNs were located in the medium-sized vessel layer. Overall, NPY(+) and TH(+) ICNs were more frequent in the central temporal area, both in isolation and forming microganglia. We also detected small spindle elements intensely immunoreactive to TH(+) and distributed mainly in the suprachoroid from the equator to the periphery. In conclusion, the human choroid contains abundant NPY and TH nerve fibers related to chroroidal vascular structures; it further possesses NPY(+) and TH(+) ICNs which contribute to the choroidal self-regulation persisting after sympathetic denervation. Additionally, these ICNs may at least partially explain why the choroidal blood flow does not respond to the factors that influence systemic vascular control. The preferential location of these cells in the submacular area suggests that dysfunction or degeneration of these cells may be a factor in vascular pathologies found in ocular disease, such as diabetic macular edema or age-related macular degeneration.

Cervical sympathectomy causes photoreceptor-specific cell death in the rat retina.

Changes in the regulation of the vasculature of the eye may be related to some age-related ocular diseases. We have previously shown that loss of sympathetic innervation, as can normally occur with age, resulted in substantial vascular growth of the choroid. The current study was designed to determine whether changes induced by sympathetic denervation causes significant loss of photoreceptors and increased glial cell reactivity in the retina. Sympathetic denervation was performed followed by immunohistochemistry, TUNEL staining, and protein expression analysis to investigate photoreceptor loss. There was a significant reduction (30%) in photoreceptor numbers in the sympathectomized eye. This loss was due to apoptosis, as there was over a doubling in apoptotic cell numbers after sympathectomy. This loss of photoreceptors in the sympathectomized eye resulted in a significantly reduced width of the outer nuclear layer of the retina when compared to the contralateral eye. Increased glial fibrillary acidic protein (GFAP) staining was also noted after sympathectomy in the ganglion cell layer with streaking toward the bipolar cell layer. These results suggest that loss of sympathetic innervation may cause significant changes to the physiology of the choroid.

Choroidal ganglion cells in prenatal, young, and middle-aged human donor eyes.

PURPOSE: In the current study, the appearance and development of choroidal ganglion cells (CGCs) was investigated in eyes of 18 human donors between the 13th week of gestation (wog) and 42 years of age. METHODS: The number and diameter of CGCs was evaluated in scleral and choroidal whole mounts stained for NADPH diaphorase. To demonstrate the synaptic input of the CGCs, sections were stained with antibodies against synaptophysin, vesicular acetylcholin transporter, tyrosin hydroxylase, and vesicular monoaminergic transporter 2. RESULTS: Clusters of small CGCs were first seen in the 18th wog next to the nasal and temporal long ciliary nerves. Immunohistochemistry in the 25th wog revealed 3298 and 5429 nitric oxide synthase/NADPH diaphorase positive CGCs, surrounded by numerous cholinergic and aminergic boutons. The number of CGCs decreased to 1000-2500 after birth. During postnatal development, the CGCs spread into small groups, distributed all over the choroid. The size of CGCs increased markedly up to adulthood. CONCLUSIONS: CGCs appear late during choroidal development, in parallel with the differentiation of the outer vascular layers. This coincidence might point to the primary role of the CGCs as specific vasoregulators in species with a well-developed fovea centralis.

Developmental cell death in vivo: rescue of neurons independently of changes at target tissues.

Programmed cell death is a prominent feature of neural development that is regulated by a variety of cell-cell interactions. We used the avian ciliary ganglion to dissect the relative contributions of target tissues vs. ganglionic inputs in regulating cell death. The two populations of the ciliary ganglion innervate different targets: choroid neurons innervate vasculature, whereas ciliary neurons innervate the iris and ciliary body. By counting after labeling all neurons with Islet-1 and choroid neurons with anti-somatostatin, we determined that alpha-bungarotoxin (alpha-btx) at 12.5 microg/day rescued only ciliary neurons, whereas 75 microg/day rescued both ciliary and choroid neurons. It is unlikely that alpha-btx acted by blocking nerve transmission at both targets because the choroid vasculature lacked transcripts for alpha-btx binding molecules. In addition, no inherent trophic activity could be ascribed to alpha-btx, and survival could not be attributed to differences in total trophic activity of eyes from saline vs. alpha-btx-treated embryos. In contrast, the alpha7 antagonist alpha-methyllycaconitine (MLA) rescued ciliary neurons at 2.6 microg/day, whereas 26 microg/day rescued choroid neurons. Nerve terminals of ciliary neurons rescued with alpha-btx were significantly larger; however, differences in nerve terminal size or branching of axons were not observed in ciliary neurons rescued with MLA or choroid neurons rescued by either MLA or alpha-btx. Our results suggest that neuronal survival can be promoted independently of changes at the target tissues when orthograde signals acting by means of neuronal alpha7 nicotinic receptors are blocked.

Neuropeptide Y and the ocular innervation of rat, guinea pig, cat and monkey.

By the immunohistofluorescence technique, peripheral nerves of the rat, guinea pig, cat and monkey eye contain a neuropeptide Y-like immunoreactive peptide. A broad distribution of immunoreactive nerve fibers is present in all four animals, innervating tissues of the aqueous humor outflow apparatus, the limbal blood vessels, and uveal blood vessels. A dense plexus of neuropeptide Y-like immunoreactive nerve fibers is present to the ciliary processes. A rich innervation exists to the iris dilator muscle, but that to the iris sphincter is modest. Throughout all regions of the uvea, neuropeptide Y-like immunoreactive nerves are associated closely with melanocytes. When acid extracts of anterior uvea and choroid from rat and guinea pig are analyzed by radioimmunoassay and reverse phase high performance liquid chromatography, the immunoreactive ocular peptide occurs in a single molecular form indistinguishable from porcine neuropeptide Y. The present findings indicate that neuropeptide Y is present in ocular nerves of rat, guinea pig, cat, and monkey. Their distribution, with a few small exceptions, closely parallels that of ocular adrenergic nerves as revealed by histofluorometric techniques. While no ocular effects of neuropeptide Y have been reported to date, its other known biological effects imply potential functions in the eye.

Cholinergic neurons of the chicken ciliary ganglion contain somatostatin.

Somatostatin immunoreactivity was studied in the avian ciliary ganglion by immunocytochemistry and radioimmunoassay. Immunoreactivity was localized to small diameter cell bodies of neurons from embryos, newly-hatched and adult preparations. Immunostaining of ganglia with a mixture of antisera to substance P and monoclonal antibody to somatostatin indicated that a number of somatostatin-immunoreactive neurons were surrounded by substance P-immunoreactive boutons, which characteristically terminate on choroidal neurons. Staining with a mixture of antisera to choline acetyltransferase and antibody to somatostatin showed that the somatostatin-immunoreactive neurons were less intensely-stained for choline acetyltransferase than were the neurons lacking somatostatin immunoreactivity. Bundles of nerve fibers showing somatostatin and choline acetyltransferase immunoreactivity were found in the choroid layers of the eye. Radioimmunoassay indicated the presence of somatostatin immunoreactivity in both chick and quail ganglia; the somatostatin immunoreactivity eluted from high pressure liquid chromatography in the same positions as authentic somatostatin 14 and 28. These results show that somatostatin is contained in cholinergic choroidal neurons in the chick and quail ciliary ganglion.

Control of choroidal blood flow by the nucleus of Edinger-Westphal in pigeons: a laser Doppler study.

Anatomical studies in birds have suggested that choroidal blood flow may be regulated by a circuit involving the following serially-connected components: the retina-the suprachiasmatic nucleus (SCN)-the medial subdivision of the nucleus of Edinger-Westphal (mEW)-the ciliary ganglion-the choroidal blood vessels. In order to better clarify the role of this circuit, we examined the effects of electrical stimulation of EW on choroidal blood flow in the ipsilateral eye, using laser Doppler velocimetry to monitor choroidal blood flow in the superior pole of the eye. Baseline choroidal blood flow values (144-311.3 mg/min per eye) were found to be comparable to those previously reported in rabbits, cats and primates. Stimulation of EW dramatically increased choroidal blood flow. The increases were current-related and the average maximal increases ranged between 300-700% above baseline values. In contrast, EW stimulation had little or no effect on overall bodily blood flow. All EW stimulation sites were later verified histologically. These results indicate that the SCN-mEW circuit in birds may be involved in mediating increases in choroidal blood flow, possibly in response to the levels of retinal illumination. Such adaptive neural regulation of choroidal blood flow may play an important role in mitigating the potentially deleterious effects of light on the retina.

Association of a choroidal ganglion cell plexus with the fovea centralis.

PURPOSE: After recently demonstrating an NADPH-diaphorase-, nitric oxide synthase (NOS)-positive ganglion cell plexus in the human choroid that was absent in rabbit and rat eyes, the authors extended their comparative studies to nonhuman primates and to subprimate mammals. METHODS: The authors investigated the choroids of diurnal cynomolgus monkeys with well-developed fovea centralis and accommodative systems; diurnal tree shrews without a fovea centralis or accommodative capacity; nocturnal owl monkeys with substantial accommodative capacity but without a fovea centralis; cats with an area centralis but no fovea centralis; and pigs without an area centralis or a fovea centralis. The latter two species have moderately developed ciliary muscles. Wholemounts of the choroid of eight cynomolgus monkey, two owl monkey, four tree shrew, four cat, and four pig eyes were stained for NADPH-diaphorase. In addition, frozen sections through the cynomolgus monkey choroid were stained for NOS and vasoactive intestinal polypeptide (VIP). RESULTS: In all species, the choroidal vessels were surrounded by NADPH-diaphorase-positive nerve fibers. A ganglion cell plexus, however, was seen only in cynomolgus monkey eyes. The ganglion cells stained for NOS and VIP. CONCLUSIONS: The presence of intrachoroidal nitrergic nerve cells restricted to species with a fully developed fovea centralis may indicate a functional correlation of these structures.

Vasoactive intestinal polypeptide and the ocular innervation.

The indirect immunofluorescence technique with antisera to vasoactive intestinal polypeptide (VIP) stains peripheral nerve fibers in both the anterior segment and the posterior segment of rat, guinea pig, cat, and rhesus monkey eyes. While immunoreactive corneal nerves are lacking, all four species have a prominent innervation of the superficial limbal blood vessels. The aqueous humor outflow apparatus of the rat, guinea pig, and cat, but not the monkey, contain VIP-like immunoreactive nerves. All four animals have immunoreactive iris nerve fibers, tending either to surround large blood vessels or to lie as free stromal nerves. Only in the cat are immunoreactive nerve fibers seen within the iris muscles. A modest number of VIP-like immunoreactive nerves are present in the ciliary body of all four animals; immunoreactive nerve fibers within the ciliary processes occur only in the rat and guinea pig. VIP-like immunoreactive nerves are found in the choroid of all four animals. An association of immunoreactive nerve fibers to uveal melanocytes also is apparent. The present findings expand several prior immunohistochemical studies of mammalian eyes in which the VIP-like immunoreactive nerves to the choroid was emphasized.

Localization of preganglionic neurons that innervate choroidal neurons of pterygopalatine ganglion.

PURPOSE: The pterygopalatine ganglion (PPG) receives preganglionic input from the superior salivatory nucleus (SSN) of the facial motor complex and is the main source of parasympathetic input to the choroid in mammals. The present study was undertaken to determine in rats the location and neurotransmitters of SSN neurons innervating those PPG neurons that target the choroid and to determine the location and neurotransmitters of the PPG choroidal neurons themselves. METHODS: Retrograde labeling from rat choroid using a fluorescent tracer, in combination with immunofluorescence labeling for nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), and choline acetyltransferase (ChAT), was used to characterize the location and neurotransmitters of choroidal PPG neurons. To identify SSN neurons that innervate the choroidal PPG neurons, the Bartha strain of the retrograde transneuronal tracer pseudorabies virus (PRV-Ba) was injected into rat choroid, and immunolabeling for NOS or ChAT was used to characterize their neurochemistry. RESULTS: Fluorescent retrograde labeling showed that PPG neurons projecting to the choroid contained NOS, VIP, and ChAT and were widely distributed in PPG and its preganglionic root, the greater petrosal nerve. SSN neurons were ChAT(+), and a subset of them was found to contain NOS. PRV-Ba transneuronal retrograde labeling revealed that choroidal preganglionic neurons were localized to the rostral medioventral part of the ipsilateral SSN. The choroidal SSN neurons were ChAT(+) and appeared largely to correspond to the NOS(+) neurons of the SSN. CONCLUSIONS: These results show that preganglionic neurons in rats that are presumed to regulate choroidal blood flow through the PPG reside within the rostral medioventral SSN, and that NOS is a marker for these SSN neurons.

The effects of choroidal or ciliary nerve transection on myopic eye growth induced by goggles.

PURPOSE: To determine the role of the choroidal and ciliary nerves and the functions they control, choroidal blood flow (CBF) and accommodation-pupil diameter, respectively, in myopia induced by form-vision deprivation. METHODS: Three groups of chicks were studied: chicks with choroidal nerves cut in the right eye, chicks with ciliary nerves cut in the right eye, and sham control chicks that received the same surgical preparation but no nerve cuts. A plastic, dome-shaped goggle was glued over the right eye of birds in all three groups after orbital surgery, and, 2 weeks later, CBF was measured using laser Doppler flowmetry. Refractive status was then measured using streak retinoscopy, and axial, nasotemporal, and dorsoventral lengths were measured using vernier calipers after enucleation. The eyes were also weighed. RESULTS: In the sham control birds, considerable ocular enlargement in all dimensions and a high degree of myopia (-14.68 diopters) was observed in the goggled eye, and CBF in the goggled eye was 66% of that in the nongoggled eye. In birds with choroidal nerve cuts, the degree of enlargement of the goggled eye was less in all dimensions, and the myopia in the goggled eye (-4.74 D) was attenuated compared to that observed in the sham controls. CBF in the goggled eye was 21% of that in nongoggled eye. Finally, in the birds with ciliary nerve cuts, nasotemporal and dorsoventral enlargement of the goggled eye were similar to that in the shams, but the axial elongation and the degree of myopia (-9.57 D) were less than observed in sham control eyes. As in the shams, CBF in the goggled eye was reduced to 59% of that in the nongoggled eye. CONCLUSIONS: These results show that although elimination of accommodation and severe reductions in CBF do affect eye growth (the latter more so), they do not prevent form-vision deprivation-induced myopia. Thus, either the mechanism of visual deprivation-induced myopia is different from that in idiopathic human myopia, or CBF levels and accommodation do not play a major role in either.