Anatomical and functional evidence for progressive age-related decline in parasympathetic control of choroidal blood flow in pigeons.

The choroid receives extensive parasympathetic innervation, which in birds arises largely from the ciliary ganglion (CG). Since age-related changes in parasympathetic regulation of choroidal blood flow (ChBF) could contribute to age-related retinal decline, we used anatomical and functional methods to determine if ChBF control by the CG shows age-related decline in pigeons. The efficacy of the choroidal vasodilatory response to activation of the CG preganglionic input from the medial subdivision of the nucleus of Edinger-Westphal (EWM) was assessed using laser Doppler flowmetry (LDF). The EWM receives bisynaptic retinal input, and electrical stimulation of EWM or light stimulation of the retina in young animals produces dramatic choroidal vasodilation. Transcleral LDF was therefore used to measure both basal ChBF and the increases in ChBF elicited by electrical stimulation of EWM or by retinal illumination in 0.5-18 year old pigeons. Fixed cryostat sections of the eye from 0.5 to 22 year old pigeons were immunolabeled for the 3A10 neurofilament-associated antigen to determine if intrachoroidal nerve fibers arising from CG exhibited age-related loss. We focused on superior choroid, since it is the primary target for CG nerve fibers. There was a marked age-related loss in the ChBF vasodilatory response elicited by either EWM stimulation or retinal illumination, as was also true for basal ChBF. A progressive decrease in choroidal nerve fibers of CG origin, to 17% of youthful abundance by 22 years of age, was also observed. The evoked ChBF increase, and basal ChBF, achieved 50% of their age-related decline between the ages of 3 and 4 years, while half the loss in CG innervation of choroid was later, occurring by 10 years. Age-related loss of choroidal nerve fibers occurs in parallel with but more slowly than the reduction in basal ChBF and the choroidal vasodilation that can be elicited via natural (light) or electrical activation of the central neural input to CG choroidal neurons. The prominent age-related decline in parasympathetic control of ChBF early in the pigeon life span could contribute to the age-related retinal decline observed in pigeons.

Choroidal blood flow in pigeons compensates for decreases in arterial blood pressure.

While it had once been thought that choroidal blood flow (ChBF) does not compensate for changes in perfusion pressure, recent studies have shown that ChBF in rabbits and humans does compensate for changes in arterial blood pressure (ABP) and thereby remains relatively stable within a physiological range of ABPs. In this study, we sought to determine if ChBF in birds can compensate for decreases in ABP, either spontaneously occuring or caused by blood withdrawal. ChBF was continuously monitored using laser Doppler flowmetry in anesthetized pigeons, and at the same time ABP was measured via the brachial artery. In studies of spontaneous fluctuation in ABP, ChBF and ABP were analyzed at regular intervals over a 2-3 hr period, while for blood withdrawal studies, blood was transiently withdrawn via the brachial artery. In both paradigms, ChBF remained near baseline over an ABP range from basal (about 90 mmHg) to about 55 mmHg, followed ABP nearly linearly below 50 mmHg, and showed no compensation below 40 mmHg. The blood withdrawal studies further showed that the compensation was more rapid with small acute declines in ABP than with larger declines. These findings reveal that ChBF in pigeons, as in rabbits and humans, compensates for declines in ABP so as to remain relatively stable within a physiological range of ABPs. Given the phylogenetic distance between humans and rabbits on one hand and birds on the other, these results suggest that choroidal compensation for ABP declines may be a common ocular mechanism among warm-blooded vertebrates.

Screening for cerebroprotective agents using an in vivo model of cerebral reversible depolarization in awake rats.

The need to screen cerebroprotective compounds without anesthetic interference prompted the development of a model using hypoxic rats. In this model two outcome measures were used: (1) the time to reach isoelectric electroencephalogram (iEEG), caused by nitrogen gas inhalation in the test chamber, and (2) the time for behavioral recovery measuring the latency of restoration of the head-withdrawal reflex upon vibrissae stimulation. We report here data of blood chemistry, cerebral tissue oxygen measurements, a definition of a proposed scoring system, and the pharmacological results of RGH-2202. The findings with RGH-2202 are used here to show the utility of the screening method. Events during hypoxia: Arterial and venous pO(2), pCO(2), and pH, and brain tissue pO(2)significantly declined. Significant correlations were established among the pO(2)of cerebral tissue, blood, and the test chamber. RGH-2202 significantly and dose-dependently shortened the iEEG time; the compound's Effective Dose(30)was 227.8 mg kg(-1). Events during recovery: Immediately after the iEEG, when the atmosphere in the chamber was replaced with room air, the arterial, venous and brain tissue pO(2)increased above the control level and subsequently recovered to baseline levels. Behavioral recovery occurred before blood chemistry was otherwise normalized. RGH-2202 significantly and dose-dependently shortened the recovery time; the Effective Dose(30)was 8.71 mg kg(-1). The available data define and support the physiological basis of this practicable rat-screening model.

Functional and morphological assessment of age-related changes in the choroid and outer retina in pigeons.

We sought to determine if choroidal and outer retinal deterioration occur with age in pigeons, as they do in other species, and investigated the relationship between age-related retinal and choroidal changes. In 64 pigeons ranging in age over the pigeon lifespan (0.5-20 years), we measured some or all among the following parameters: choroidal blood flow (ChBF) by laser Doppler flowmetry, choroidal thickness and choriocapillary vessel abundance by LM histology, choriocapillary endothelial cell transport specializations by EM histology, acuity by behavioral methods, and degenerating photoreceptor abundance and total photoreceptor abundance by LM histology. Regression and Receiver Operator Curve (ROC) analyses were used to characterize the pattern of age-related changes and determine the ages at or by which significant changes occurred. For the 45 birds for which we measured choroidal parameters, choriocapillary vessel abundance showed a curvilinear decline with age and half of this decline occurred by 3.5-4.6 years. The endothelial cell transport specializations called channels also declined curvilinearly with age. Choroidal thickness was slightly increased between the ages of 3-6 years, and thereafter declined steadily so that choroidal thickness in the oldest birds was half that in the youngest. ChBF showed an abrupt decline of about 20% at 4 years and a further 20% decline thereafter. In the 53 birds for which we obtained visual acuity and/or photoreceptor data, we observed a curvilinear decline in acuity (with half the decline having occurred by 8 years) and a prominent stepwise decline of about 20% in photoreceptor abundance at 4.7 years, followed by further decline thereafter. The period of major photoreceptor loss coincided with ages during which about 10% of photoreceptors appeared to show degenerative changes (4-8 years of age). Using partial correlation analysis with the common effect of age held constant, ChBF was found to have a positive correlation with acuity. Our results show that ChBF and choroidal vascularity decline significantly with age in pigeons, as do acuity and photoreceptor abundance. Our statistical analyses suggest that prominent choroidal vascular decline preceded the visual decline, and that there is a positive relationship between choroidal and visual functions. Thus, our findings are consistent with the view that age-related decline in choroidal function might contribute to age-related vision loss in pigeons.

Cerebroprotective drugs shorten the hypoxia-induced onset of electrical silence in unanesthetized rats.

Pharmacological agents that delay the hypoxic arrest of neuronal electrical activity, as indicated by the suppression of electroencephalogram (EEG), have previously been thought to increase brain resistance to oxygen insufficiency. On the other hand, acceleration of the EEG suppression may offer some protection against severe hypoxia by reducing neuronal energy spending on electrogenesis. In unanesthetized rats we examined the effects of several antihypoxic drugs on the time of appearance of isoelectric EEG (tiEEG), caused by normobaric hypoxia. In addition, alterations in cerebral blood flow induced by hypoxia and by some drugs were monitored using polarographic techniques to determine if cerebrocirculatory changes play a significant role in the drug effects on tiEEG. We also assessed drug effects on behavioral recovery after hypoxia by measuring the latency of restoration of the head-withdrawal reflex upon vibrissae stimulation. Pentobarbital (30 and 60 mgkg(-1)i.p.), chloralhydrate (400 mgkg(-1)i.p.) flunarizine (50-100 mgkg(-1)p.o.), hydergine (3-50 mgkg(-1)p.o.), nicergoline (50 mgkg(-1)and 85 mgkg(-1)p.o.), sabeluzole (3 and 7.5 mgkg(-1)i.p.) and vincamine (80 mgkg(-1)p.o.) reduced tiEEG (mean 27.1 +/- 3.3 min prior to drugs). In contrast, idebenone (29-85 mgkg(-1)p.o.) and vinpocetine (29-85 mgkg(-1)p.o.) had no significant effects on tiEEG. The divergent effects on cerebral blood flow suggest an insignificant role for cerebrocirculatory changes in the drug-induced reduction of tiEEG during severe hypoxia. The drug effects on recovery of the head-withdrawal reflex (mean 4.2 +/- 1.3 min prior to drugs) varied from a delay (sabeluzole) to acceleration (flunarizine) with no correlation to the effects on tiEEG, suggesting that EEG criteria alone may not predict the course of functional recovery.

Role of muscarinic cholinergic transmission in Edinger-Westphal nucleus-induced choroidal vasodilation in pigeon.

Activation of the parasympathetic ciliary ganglion input to the choroid causes increases in choroidal blood flow. We examined the role and the type of muscarinic receptors within the choroid that are involved in these increases in choroidal blood flow, using electrical stimulation of the nucleus of Edinger-Westphal (EW) to activate the ciliary ganglion input to choroid in ketamine anesthetized pigeons. Baseline choroidal blood flow and its EW-evoked increases measured as peak and total (area under the curve) responses were determined using laser Doppler flowmetry. The EW-evoked responses were reduced dose-dependently after administration of 4-diphenyl-acetoxy-N-methylpiperedine (4-DAMP), a relatively selective antagonist of M3 type muscarinic receptors, with a maximal mean decrease of 86% (peak response) and 93% (total response) at a dose of 10 microg kg(-1)i.v. without a significant effect on baseline choroidal blood flow, heart rate or systemic arterial blood pressure. Atropine, a non-selective antagonist of muscarinic receptors, decreased the EW-evoked responses to a lesser extent than 4-DAMP after intravenous administration of 1 mg kg(-1)(by 67% for peak response and by 53% for total response) or topical administration of a 5% solution (by 41% for peak response and by 62% for total response), both of which increased heart rate and systemic arterial blood pressure without a consistent effect on baseline choroidal blood flow. In contrast, himbacine (i.p. 10 microg kg(-1)), a relatively selective antagonist of M2 type muscarinic receptors, increased the EW-evoked parasympathetic cholinergic vasodilation (by 93% for the peak response and by 142% for the total response) without a significant effect on heart rate, systemic arterial blood pressure or baseline choroidal blood flow. The results of our study suggest a major role of M3 type muscarinic receptors in the EW-evoked increases in choroidal blood flow. Based on findings that the ciliary ganglion input to choroid does not synthesize nitric oxide but inhibitors of NO production do block EW-evoked choroidal vasodilation, it seems likely that the M3 receptors acted on by 4-DAMP are present on choroidal endothelial cells and mediate choroidal vasodilation via stimulation of endothelial release of nitric oxide. In contrast, M2 muscarinic receptors may play a presynaptic role in downregulating EW-evoked parasympathetic cholinergic vasodilation in avian choroid.

Preganglionic endings from nucleus of Edinger-Westphal in pigeon ciliary ganglion contain neuronal nitric oxide synthase.

The avian ciliary ganglion (CG) controls choroidal blood flow by its choroidal neurons, and pupil constriction and accommodation by its ciliary neurons. It was previously reported that both choroidal and ciliary neurons label positively for NADPH diaphorase (NADPHd), a marker for nitric oxide synthase (NOS). To assess if this labeling is preganglionic or postganglionic and to determine if it is attributable to neuronal NOS (nNOS), we studied pigeon CG using NADPHd histochemistry and nNOS immunohistochemistry (IHC). Short-duration staining times by NADPHd histochemistry yielded intense labeling of structures that appeared to be the cap-like endings on ciliary neurons and the boutonal endings on choroidal neurons that arise from the nucleus of Edinger-Westphal (EW), and light or no postganglionic perikaryal staining. The light postganglionic staining that was observed tended to be localized to ciliary neurons. Consistent with this, NADPHd+ nerve fibers were observed in the postganglionic ciliary nerves but rarely in the postganglionic choroidal nerves. These same staining times yielded robust staining of neurons in the orbital pterygopalatine microganglia network, which are known to be nNOS+. Diffuse staining of CG perikarya was observed with longer staining durations, and this staining tended to mask the preganglionic labeling. Preganglionic NADPHd+ staining in CG with short staining times was blocked by the NOS inhibitors iodonium diphenyl (IDP) and dichlorophenol-indophenol (DPIP), but the diffuse postganglionic staining observed with the longer staining times was not completely blocked. Labeling of CG sections for substance P (SP) by IHC (which labels EW-originating preganglionic endings in CG) and subsequently for NADPHd confirmed that NADPHd was localized to preganglionic endings on CG neurons. Immunohistochemical double labeling for nNOS and SP or enkephalin further confirmed that nNOS is found in boutonal and cap-like endings in the CG. Two studies were then carried out to demonstrate that the nNOS+ preganglionic endings in CG arise from EW. First, NADPHd+ and nNOS+ neurons were observed in EW in pigeons treated with colchicine to enhance perikaryal labeling. Second, NADPHd+ and nNOS+ preganglionic endings were eliminated from CG ipsilateral to an EW lesion. These various results indicate that NOS is present in EW-arising preganglionic endings on choroidal and ciliary neurons in avian CG. NOS also appears to be found in some ciliary neurons, but its presence in choroidal neurons is currently uncertain.

Effects of halothane and urethane-chloralose anaesthesia on the pressor and cerebrovascular responses to 7-NITROINDAZOLE, an inhibitor of nitric oxide synthase.

We examined the effect of 7-nitroindazole (7NI), a reportedly relatively specific inhibitor of the neuronal isoform of nitric oxide synthase (nNOS), on mean arterial blood pressure and on cerebral blood flow in rats under three different types of anaesthesia: urethane-chloralose, halothane, or urethane preceded by induction of anaesthesia with halothane. In rats under urethane-chloralose anaesthesia, 7NI induced an increase in mean systemic arterial blood pressure. In contrast, halothane used for induction and maintenance of anaesthesia eliminated the 7NI-induced systemic pressor effect, while halothane used only for induction of anaesthesia greatly attenuated the 7NI-induced systemic pressor effect. Cerebral blood flow, as measured by Laser Doppler flowmetry, decreased significantly to 85-72% of baseline within 5-10 min after i.p. 7NI injection regardless of the type of anaesthesia. Blockade of the systemic pressor effect of 7NI by halothane but not of the reduction in cerebral blood flow produced by 7NI is consistent with prior evidence that: (1) the cerebral vasculature and the peripheral vasculature differ in the isoforms of NOS involved in maintaining vascular tone, with nNOS more important in the former and endothelial NOS (eNOS) in the latter; and (2) halothane interferes with eNOS-mediated vascular tone but not nNOS-mediated control of cerebral blood flow. The fact that 7NI yields a pressor effect that can be attenuated by halothane, as also true for isoform-non-selective NOS inhibitors, raises the possibility that 7NI may to some extent inhibit endothelial NO formation. (c) 1998 The Italian Pharmacological Society.

Innervation of orbital and choroidal blood vessels by the pterygopalatine ganglion in pigeons.

Orbital and choroidal blood vessels in mammals are known to receive a parasympathetic innervation from the pterygopalatine ganglion, which appears to utilize vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) to increase choroidal blood flow. The present studies were undertaken to elucidate the anatomical and neurotransmitter organization of the pterygopalatine ganglion input to orbital and choroidal blood vessels in pigeons. Single- or double-label immunohistochemistry were employed on paraformaldehyde-fixed cryostat sections of the pigeon eye and surrounding orbital tissue to localize 1) VIP+ neurons and fibers; 2) choline acetyltransferase (CHAT)-containing cholinergic neurons and fibers; 3) axons containing the 3A10 neurofilament-associated antigen; and 4) neuronal NO synthase (nNOS)-containing neurons and fibers. NOS+ neurons and fibers were also identified by NADPH-diaphorase histochemistry in sections and whole-mount specimens. The pterygopalatine ganglion was found to consist of an interconnected series of three to four main microganglia of about 50-200 neurons each and numerous lesser microganglia. The major microganglia of the pterygopalatine network in pigeon lie along the superior aspect of the Harderian gland, with many additional fibers and microganglia of the network encircling the gland. Neurons of all microganglia were extremely rich in VIP, nNOS, and NADPH-diaphorase and moderate in CHAT. The majority of the pterygopalatine ganglion neurons were observed to co-contain VIP and nNOS. Axons labeled for VIP, nNOS, NADPH-diaphorase, or the 3A10 antigen could be traced from the pterygopalatine ganglion network to perivascular fiber plexi on orbital blood vessels. These orbital vessels, many of which enter the choroid posteriorly and nasally, appear to be a conduit by which pterygopalatine postganglionic fibers reach the choroid. The pterygopalatine postganglionic fibers were also seen to innervate the Harderian gland and contribute branches to the nearby ophthalmic nerve. Within the choroid, VIP+ fibers were widely scattered and sparse but were most abundant in nasal choroid. A few VIP+ and NADPH- diaphorase+ neurons were also observed in the choroid. These results suggest that pterygopalatine ganglion neurons of birds use VIP and NO to exert vasodilatory control over blood flow to and within the avian choroid.

Central neural circuits for the light-mediated reflexive control of choroidal blood flow in the pigeon eye: a laser Doppler study.

Electrical stimulation in pigeons of the input from the medial subdivision of the nucleus of Edinger-Westphal (EWM) to the choroidal neurons of the ipsilateral ciliary ganglion, which themselves have input to the choroidal blood vessels of the ipsilateral eye, increases choroidal blood flow (ChBF). Since the EWM receives input from the contralateral suprachiasmatic nucleus (SCN), which in turn receives contralateral retinal input, the present study sought to determine if activation of the SCN by microstimulation or by retinal illumination of the contralateral eye would also yield increases in ChBF in that same eye. Using laser Doppler flowmetry (LDF) to measure ChBF, we found that electrical activation of the contralateral SCN by 100-Hz anodal pulse trains yielded increases in ChBF that were stimulus related and proportional to the stimulating current. These increases in ChBF elicited by the SCN stimulation were accompanied by increases in choroidal volume (vasodilation), but not by increases in systemic blood pressure. Furthermore, the increases could be blocked reversibly by lidocaine injection into the EWM. These results suggest that the increases in ChBF in the eye contralateral to the SCN stimulation were specifically mediated by the SCN-EWM pathway. Retinal illumination with a fiber optic light source was also found to increase ChBF in the illuminated eye, and these effects too could be blocked reversibly with lidocaine injection into the EWM or permanently by the EWM lesion. Control studies confirmed that the light-elicited increases were mediated by increases in choroidal volume (i.e. vasodilation), were not accompanied by systemic blood pressure increases, and were not artifactually generated by transocular illumination of the LDF probe. Thus, the SCN-EWM circuit may be involved in regulating ChBF in response to the level of retinal illumination and/or the visual patterns falling on the retina.

Evidence from its cardiovascular effects that 7-nitroindazole may inhibit endothelial nitric oxide synthase in vivo.

We have examined whether the cardiovascular effects of 7-nitroindazole, a reportedly selective inhibitor of neuronal nitric oxide (NO) synthase, are induced without inhibition of endothelial NO synthase. A significant increase in mean arterial blood pressure but no change in heart rate was observed after 7-nitroindazole administration (50 mg/kg i.p.) in rats anesthetized with urethane or urethane and chloralose, while both an elevation in mean arterial blood pressure and bradycardia were observed in conscious animals after 7-nitroindazole administration (50 mg/kg i.p.). No enhancements in these effects on mean arterial blood pressure and heart rate were observed in urethane-chloralose anesthetized rats treated with a higher dose of 7-nitroindazole (75 mg/kg i.p.). Use of halothane to induce anesthesia abolished the pressor effect of 7-nitroindazole in rats studied under urethane anesthesia. 7-Nitroindazole shortened the duration of the acetylcholine (3 micrograms or 30 micrograms i.v.) but not the sodium nitroprusside (2 micrograms i.v.) induced hypotension in urethane-anesthetized rats. Pretreatment with L-arginine (300 mg/kg i.v.) inhibited the effects of 7-nitroindazole on mean arterial blood pressure and acetylcholine induced hypotension, suggesting involvement of the L-arginine-NO pathway in the effects of 7-nitroindazole. The effects of 7-nitroindazole on blood pressure and on the depressor responses to acetylcholine and sodium nitroprusside are similar to the effects previously observed after non-selective NO synthase inhibition by L-arginine analogs. Our results suggest, therefore, that 7-nitroindazole affects basal endothelial NO formation in vivo. The suppressive action of halothane on the cardiovascular effects of 7-nitroindazole suggests that the influence of anesthetics should be taken into consideration in studies of the cardiovascular effects of NO synthase inhibitors, particularly 7-nitroindazole.

Neural nitric oxide mediates Edinger-Westphal nucleus evoked increase in choroidal blood flow in the pigeon.

PURPOSE: Nitric oxide (NO) has been identified as a putative neurotransmitter in choroidal perivascular nerve fibers originating parasympathetically. Although constitutively produced NO has been implicated in the regulation of the choroidal circulation, the specific role of neurally derived NO in choroidal vasodilation has not been determined. This study examined the role of neurally derived NO in the control of the choroidal blood flow (ChBF) in vivo. METHODS: Resting ChBF and a increase in ChBF elicited by electrical stimulation of the nucleus of Edinger-Westphal (EW) were measured transclerally by laser Doppler flowmetry in anesthetized pigeons before and after administration of a selective inhibitor of neural NO synthase, 7-Nitroindazole (7NI; 50 mg/kg given intraperitoneally); a nonselective NO synthase inhibitor, Ng-nitro-L-arginine methyl ester (L-NAME; 30 mg/kg given intravenously); L-arginine (300 mg/kg given intravenously) followed by 7NI (50 mg/kg given intraperitoneally); or vehicle. RESULTS: The 7NI and L-NAME, but not the vehicle, attenuated the EW-evoked response (maximally by 78% and 83%, respectively), and this effect lasted for at least 1 hour. Pretreatment with L-arginine abolished this effect of 7NI. Resting ChBF was reduced and systemic blood pressure was increased after L-NAME administration, but both were unchanged after 7NI or vehicle were administered. CONCLUSIONS: Neurally derived NO is responsible for a major component of the ChBF increase caused by EW stimulation in pigeons. This represents the first demonstration in vivo that neuronally produced NO is an important factor in the control of ChBF by the parasympathetic nervous system. In particular, neuronally produced NO appears to play a role in rapid upregulation of ChBF in the pigeon, whereas endothelially produced NO plays a major role in control of resting ChBF.