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.

Evidence that urocortin is absent from neurons of the Edinger-Westphal nucleus in pigeons.

The Edinger-Westphal nucleus (EWN) is a central preganglionic parasympathetic cell group that gives rise to cholinergic input to the ciliary ganglion, thereby regulating several neurovegetative ocular functions. Recently, the supposed presence of the neuropeptide urocortin (UCN) has been reported in EWN neurons in rodent brain. The purpose of the present study was to examine the distribution of UCN in avian brain and to investigate by immunohistochemical analysis the possible use of this substance as an EWN marker in a non-mammalian class of vertebrates. Brain tissue of pigeons was incubated with a specific antibody against UCN and the results showed labeling of many small neurons, forming a double wing in the dorsal mesodiencephalic transition area. Their size and shape, however, differed from those of EWN neurons, and they were preferentially located rostral to the EWN. Double-label experiments employing an antibody against the enzyme choline acetyltransferase (ChAT) showed that UCN is not localized to the cholinergic cells of the EWN and confirmed the rostral distributionof UCN never overlapping the ChAT+ EWN cells. Taken together, these results suggest that, at least in pigeons, the UCN+ population does not belong to the traditionally defined EWN.

Evidence that urocortin is absent from neurons of the Edinger-Westphal nucleus in pigeons

The Edinger-Westphal nucleus (EWN) is a central preganglionic parasympathetic cell group that gives rise to cholinergic input to the ciliary ganglion, thereby regulating several neurovegetative ocular functions. Recently, the supposed presence of the neuropeptide urocortin (UCN) has been reported in EWN neurons in rodent brain. The purpose of the present study was to examine the distribution of UCN in avian brain and to investigate by immunohistochemical analysis the possible use of this substance as an EWN marker in a non-mammalian class of vertebrates. Brain tissue of pigeons was incubated with a specific antibody against UCN and the results showed labeling of many small neurons, forming a double wing in the dorsal mesodiencephalic transition area. Their size and shape, however, differed from those of EWN neurons, and they were preferentially located rostral to the EWN. Double-label experiments employing an antibody against the enzyme choline acetyltransferase (ChAT) showed that UCN is not localized to the cholinergic cells of the EWN and confirmed the rostral distributionof UCN never overlapping the ChAT+ EWN cells. Taken together, these results suggest that, at least in pigeons, the UCN+ population does not belong to the traditionally defined EWN.

Nursing care for raised intra-abdominal pressure and abdominal decompression in the critically ill.

Abdominal assessment is one of a number of continuous assessments that critical care nurses undertake. Since 1988 in the Department of Critical Care Medicine (DCCM), the technique of abdominal decompression has become another therapy for severe critical illness. The critical care nurse requires to have an understanding of raised intra-abdominal pressure assessment, pressure measurement and the care of abdominal polypropylene mesh insertion in the critical care setting. Our experience has been that the use of polypropylene mesh insertion halved since 1993. A retrospective study (Torrie et al. 1996) of 68 occasions (64 patients) of polypropylene mesh insertion, showed that seven patients developed fistulas and 32 patients died. There was no dehiscence of the mesh from the fascia. Forty-two wounds had primary fascial closure (28 with primary skin closure, 3 with secondary skin closure, 11 left to granulate) and 3 of them later dehisced. At follow-up (27 patients, median 7.5 months), 6 had stitch sinuses, and 5 had incisional hernias. Care of patients with polypropylene mesh inserted requires vigilant nursing practice but decompression of raised intra-abdominal pressure can be life-saving and complications are manageable.

Bereavement follow-up after critical illness.

OBJECTIVE: To describe the establishment and initial activity of a Bereavement Follow-up Service for next-of-kin of patients who died in an intensive care unit (ICU) and to quantify aspects of their experience including quality of intensive care service and the early impact on next-of-kin of their bereavement. DESIGN: Cross-sectional prospective study, which was conducted by a structured telephone interview. SETTING: A 14-bed adult general ICU in a tertiary university hospital. SUBJECTS: A total of 99 next-of-kin of patients who died in ICU. INTERVENTIONS: Referral to other agencies if requested. MEASUREMENTS AND MAIN RESULTS: Attempts were made to contact the next-of-kin of all 151 patients who died in 1995, and 104 were contacted. Five declined to be interviewed. The results refer to 99 who consented to telephone interview a median of 33 days after the death. A total of 84 considered themselves well informed during the intensive care period, 76 understood the fatal sequence of events but 19 of them would have liked more information. A total of 77 had positive comments about the quality of care, most commonly about compassionate behavior (58), but 30 had negative comments, most commonly about poor communication (13). Only 7 were living alone, 85 had resumed normal activities, 40 of 47 workers had returned to work, 58 had sleep disturbance at some stage (still present in 44), but only 12 were taking sedatives or antidepressants. A total of 32 had financial difficulties and 21 were referred to other agencies, most commonly grief counselors. CONCLUSIONS: We were disappointed to contact only two thirds of next-of-kin, but results from these subjects demonstrated a high level of satisfaction with the care given. Nevertheless, some were dissatisfied with the quality of service they experienced. Most had resumed their normal activities, including work, and few were living alone. However, sleep disturbance and financial difficulty were common, and some requested help from other support agencies.

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.

Influence of ophthalmic nerve fibers on choroidal blood flow and myopic eye growth in chicks.

Ophthalmic sensory nerve fibers containing substance P and calcitonin gene-related peptide' innervate the choroid in mammals and are known to vasodilate choroidal blood vessels. The avian choroid is also innervated by ophthalmic nerve fibers containing substance P and calcitonin gene-related peptide. The present studies were carried out to determine the influence of these sensory fibers on choroidal blood flow in birds and characterize their interaction with manipulations affecting eye growth. In these studies, ChBF was measured using laser Doppler flowmetry in both eyes in the following groups of birds: (1) normal chicks; (2) chicks with right optic nerve transected for 2 weeks; (3) chicks with right optic nerve transected and a goggle over the right eye for 2 weeks; and (4) chicks with right optic and ophthalmic nerves transected and a goggle over the right eye for 2 weeks. The eyes were refracted and various ocular dimensions measured after the blood-flow measurements. It was found that optic nerve transection reduced ChBF to 30% of normal. Placing a goggle (which increases ocular temperature by 4 degrees C) over an optic nerve transected eye nearly doubled choroidal blood flow over that in an optic nerve transected eye without a goggle. Additional transection of the ophthalmic nerve in a goggled optic nerve-transected eye, yielded choroidal blood flow that was indistinguishable from that in a nongoggled optic nerve-transected eye. Optic nerve transection had a slight stunting effect on axial growth of the eye. While myopic axial elongation was observed in goggled eyes with the optic nerve cut, the extent of myopia was less than in normal goggled eyes. Ophthalmic nerve transection further reduced the myopia induced by goggling in an optic nerve cut eye. These results suggest that ophthalmic nerve input to the choroid exerts a vasodilatory influence, which is activated in a goggled eye. This increased choroidal blood flow may be in response to elevated ocular temperatures caused by the goggling and this increase appears to be masked in goggled eyes with an intact optic nerve by the reduction in choroidal blood flow normally accompanying myopic eye growth. Our results thus show that the induction of myopic eye growth (as in our optic nerve cut eyes with a goggle) need not be accompanied by a decrease in choroidal blood flow from the baseline no-goggle condition (in this case, with the optic nerve cut).

A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections.

The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohistochemical studies. Enzymatic digestion, formic acid treatment, microwave heating and autoclave heating have been used to deal with this problem, with microwave heating-based antigen retrieval having become widely used as the method of choice. Microwave heating, however, has the shortcoming that it is difficult to precisely control the heating temperature and it is difficult to apply this method of heating to free-floating sections without damaging the sections. We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating or paraffin-embedded slide-mounted sections. We found that the best results were obtained with a 30 min incubation in a 10-50 mM sodium citrate solution (pH 8.5-9.0) preheated to and maintained at 80 degrees C in a water-bath, followed by 30 min incubation in 0.3-3% nonfat dry milk to reduce nonspecfic staining. This method is highly effective for both 40 microm free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections, and it works well for tissue from diverse species (human, rat, mouse, pigeon, and zebra finch) and for diverse antigens (e.g. enkephalin, substance P, huntingtin, GluR1, GFAP, and ubiquitin). This method was also found to enhance immunolabeling in glutaraldehyde-fixed tissue that had been prepared for ultrastructural examination, without having a deleterious effect on the ultrastructure.

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.

Distribution within the choroid of cholinergic nerve fibers from the ciliary ganglion in pigeons.

The distribution of the ciliary ganglion (CG) innervation to the pigeon choroid was determined immunohistochemically, using antisera against choline acetyltransferase (CHAT) and a neurofilament-related protein (the 3A10 antigen). Single-labeling revealed that the nerve fibers containing these two antigens were similarly distributed in the pigeon choroid, with the superior and temporal quadrants of the eye containing the most fibers. Both types of fibers surrounded and ramified on choroidal blood vessels. Additionally, CHAT+ varicosities were evident among vessels in the choroid and choriocapillaris. Double-label immunofluorescence revealed that CHAT and the 3A10 antigen were almost completely colocalized in choroidal nerve fibers, but absent from CHAT+ varicosities. Substance P-containing and calcitonin gene-related peptide-containing choroidal nerve fibers were poor in 3A10+ labeling. Transection of the postganglionic fibers of the CG reduced CHAT+ and 3A10+ nerve fibers in the choroid to 3-5% of normal abundance, with most of the residual fibers being located in the nasal and inferior quadrants. The present results suggest that the CG in pigeon preferentially influences choroidal blood flow in the superior and temporal parts of the eye, which are involved in high acuity and binocular vision.

Evidence from aequorin for injury of metabolically inhibited myocytes independently of free Ca2+.

The calcium-sensitive photoprotein aequorin has been used to measure cytoplasmic free Ca in single rat ventricle myocytes during metabolic inhibition. Rounding-up of the myocytes, and blebbing of the surface, occurred while free Ca remained at normal resting levels of 1 to 3 X 10(-7)M. Control over free Ca is less susceptible to metabolic blockade than is structural organization.