Immunohistochemical study of pig retinal development

PURPOSE: The pig eye is similar to the human eye in terms of anatomy, vasculature, and photoreceptor distribution, and therefore provides an attractive animal model for research into retinal disease. The purpose of this study was to characterize retinal histology in the developing and mature pig retina using antibodies to well established retinal cell markers commonly used in rodents.

METHODS: Eyes were enucleated from fetuses in the 9th week of gestation, 1 week old piglets and 6 months old adult animals. Eyeglobes were fixed and cryosectioned. A panel of antibodies to well established retinal markers was employed for immunohistochemistry. Fluorescently labeled secondary antibodies were used for signal detection, and images were acquired by confocal microscopy. Mouse retina at postnatal day (P) 5 was used as a reference for this study to compare progression of histogenesis. Most of the primary antibodies have previously been used on mouse tissue.

RESULTS: Most of the studied markers were detected in midgestation pig retina, and the majority had a similar distribution in pig as in P5 mouse retina. However, rhodopsin immunolabeling was detected in pig retina at midgestation but not in P5 mouse retina. Contrary to findings in all rodents, horizontal cells were Islet1-positive and cones were calbindin-immunoreactive in pig retina, as has also been shown for the primate retina. Recoverin and rhodopsin immunolabeling revealed an increase in the length of photoreceptor segments in 6 months, compared to 1 week old animals.

CONCLUSIONS: Comparison with the published data on human retina revealed similar marker distribution and histogenesis progression in the pig and human retina, supporting the pig as a valuable animal model for studies on retinal disease and repair. Furthermore, this study provides information about the dynamics of retinal histogenesis in the pig and validates a panel of antibodies that reliably detects developing and mature retinal cell phenotypes in the pig retina.

Efficacy and safety of peri-prostatic local anaesthetic injection in tram-rectal biopsy of the prostrate:A prospective randomised study

Objective: To assess the efficacy and safety of periprostatic lignocaine injection in trans-rectal ultrasound (TRUS) -guided biopsy of the prostate gland.

Methods: Ninety- six men (mean age 65 years, range 47-74) undergoing TRUS biopsy were randomised into the local anaesthetic (LA) or placebo group. Six to twelve biopsy cores were taken, the majority being 10 cores. Patients were asked to fill in the expected pain score on a visual analogue scale (VAS) prior to the procedure. They also completed the actual pain experienced on VAS after the biopsy. The incidence of complications was documented.

Results: The age, mean prostate specific antigen (PSA) were comparable in both groups. The expected pain score was also comparable (5.2 +/- 1.6 in LA, 5.0 +/- 1.4 in Placebo). In the LA group, the mean actual pain score was 3.0 +/- 1.8 and in the placebo group it was 6.5 +/- 2.2 (P = 0.0001). When patients were asked whether they would undergo the procedure again in the same way, 100% of the LA group and only 64% of the placebo group responded 'yes'(P=0.002 using Fisher's test). The complication rates were not significantly different between the two groups.

Conclusion: Peri-prostatic injection of local anaesthetic is safe and reduces discomfort significantly, and should be routinely offered to patients.

Non-invasive antidromic neurostimulation:A simple effective method for improving bladder storage

Patients with intractably diminished bladder storage function are encountered frequently by neurourologists, occasionally requiring reconstructive surgery for appropriate resolution. Although sacral neuromodulation is a recognized effective therapeutic modality, present techniques are technically demanding, invasive, and expensive. This study investigated the effect of non-invasive third sacral nerve (S3) stimulation on bladder activity during filling cystometry. One hundred forty-six patients underwent standard urodynamic filling cystometry that was then immediately repeated. Patients in the study group (n = 74) received antidromic transcutaneous sacral neurostimulation during the second fill and the control group (n = 72) underwent a second fill without neurostimulation. A statistically significant increase in bladder storage capacity without a corresponding rise in detrusor pressure was observed in the neurostimulated patients. This improvement in functional capacity is an encouraging finding that further supports the use of this non-invasive treatment modality in clinical practice. Neurourol. Urodynam. 20:73-84. 2001. (C) 2001 Wiley-Liss, Inc.

Transcutaneous sacral neurostimulation for irritative voiding dysfunction

with refractory irritative voiding dysfunction. Following an initial response, patients may successfully apply this treatment themselves to ensure long-term relief. Objective: Patients with irritative voiding dysfunction are often unresponsive to standard clinical treatment. We evaluated the response of such individuals to transcutaneous electrical stimulation of the third sacral nerve. Methods: 32 patients with refractory irritative voiding dysfunction (31 female and 1 male; mean age 47 years) were recruited to the study. Ambulatory transcutaneous electrical neurostimulation was applied bilaterally to the third sacral dermatomes for 1 week. Symptoms of frequency, nocturia, urgency, and bladder pain were scored by each patient throughout and up to 6 months following treatment. Results: The mean daytime frequency was reduced from 11.3 to 7.96 (p = 0.01). Nocturia episodes were reduced from a mean of 2.6 to 1.8 (p = 0.01). Urgency and bladder pain mean symptom scores were reduced from 5.97 to 4.89 and from 1.48 to 0.64, respectively. After stopping therapy, symptoms returned to pretreatment levels within 2 weeks in 40% of the patients and within 6 months in 100%, Three patients who continued with neurostimulation remained satisfied with this treatment modality at 6 months. Conclusions: Transcutaneous third sacral nerve stimulation may be an effective and noninvasive ambulatory technique for the treatment of patients with refractory irritative voiding dysfunction. Following an initial response, patients may successfully apply this treatment themselves to ensure long-term relief.

Effects of thyroxine treatment on histology and behavior using the methimazole model of congenital hypothyroidism in the rat

The timing of thyroxine (T4) replacement treatment in congenital hypothyroidism (CH) has been suggested to be important for optimizing cognitive recovery in humans; however this has not been fully established using modern animal models of CH. Consequently, the current studies investigated the ameliorating effects of postnatal T4 treatment on neuropathology and behavior in CH rats. Rat dams were administered methimazole to produce CH offspring, then brain tissue from male CH pups was analyzed to determine the effects of postnatal (P3, P7, P14 and P21) T4 treatment on hippocampal dendritic branching and the expression of nerve growth factor (NGF). Two operant behavioral procedures were employed to confirm and extend previous findings obtained using this model, and to investigate timelines for instigating T4 treatment on improved behavioral outcomes. T4 treatment initiated at P14 was protective of a reduction in dendritic branching in the hippocampus, and initiated at P7 was protective of a reduction of NGF expression in the fimbria of the hippocampus. Induction of CH did not affect the acquisition of simple operant response rules but had a significant effect on the acquisition of complex operant rules subsequently imposed. Furthermore, T4 treatment initiated at P3 protected learning deficits seen following the imposition of complex operant response rules. These findings indicate T4 treatment initiated at P7 is sufficient for the protection of hippocampal NGF expression and dendritic branching but for the protection of complex behavioral abilities T4 treatment is necessary prior to or approximating P3.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

Neuromusculature of Gyrodactylus rysavyi, a monogenean gill and skin parasite of the catfish Clarias gariepinus

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry in conjunction with confocal scanning laser microscopy were used for the first time to describe the nervous and muscle systems of the viviparous monogenean parasite, Gyrodactylus rysavyi inhabiting the gills and skin of the Nile catfish Clarias gariepinus. The body wall muscles are composed of an outer layer of circular fibres, an intermediate layer of paired longitudinal fibres and an inner layer of well-spaced bands of diagonal fibres arranged in two crossed directions. The musculature of the pharynx, intestine, reproductive tract and the most prominent muscles of the haptor were also described. Two characteristic muscular pads were found lying in the anterior region of the haptor in close contact with the hamuli. To each one of these pads, a group of ventral extrinsic muscles was connected. The role of this ventral extrinsic muscle in the body movement was discussed. The mechanism operating the marginal hooklets was also discussed. The central nervous system (CNS) consists of paired cerebral ganglia from which three pairs of longitudinal ventral, lateral and dorsal nerve cords arise. The nerve cords are connected at intervals by many transverse connectives. The CNS is better developed ventrally than dorsally or laterally and it has the highest reactivity for all neuroactive substances examined. Both the central and the peripheral nervous system (PNS) are bilaterally symmetrical. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts were explained. The results implicated acetylcholine, FMRFamide-related peptides (FaRPs) and serotonin in sensory and motor function. The results were compared with those of the monogeneans Macrogyrodactylus clarii and M. congolensis inhabiting the gills and skin respectively of the same host fish C. gariepinus.

Neuromusculature of Macrogyrodactylus congolensis, a monogenean skin parasite of the Nile catfish Clarias gariepinus

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry, in conjunction with confocal scanning laser microscopy, were used to describe the neuromusculature of the monogenean skin parasite Macrogyrodactylus congolensis from the Nile catfish Clarias gariepinus. The body wall muscles are composed of an outer layer of compactly arranged circular fibres, an intermediate layer of paired longitudinal fibres and an inner layer of well-spaced bands of diagonal fibres arranged in two crossed directions. The central nervous system consists of paired cerebral ganglia from which three pairs of longitudinal ventral, lateral and dorsal nerve cords arise. The nerve cords are connected at intervals by many transverse connectives. Both central and peripheral nervous systems are bilaterally symmetrical and better developed ventrally than laterally and dorsally. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts were examined. Results implicate acetylcholine, FMRFamide-related peptides and serotonin in sensory and motor function. The results were compared with those of Macrogyrodactylus clarii, a gill parasite of the same host fish C. gariepinus.

Neuromusculature of Macrogyrodactylus clarii, a monogenean gill parasite of the Nile catfish Clarias gariepinus in Egypt

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry in conjunction with confocal scanning laser microscopy have been used for the first time to describe the nervous and muscle systems of the viviparous monogenean gill parasite, Macrogyrodactylus clarii. The gross spatial arrangement of muscle and associated cholinergic, peptidergic and aminergic innervations has been examined. The central nervous system (CNS) consists of paired cerebral ganglia from which emanate three pairs of longitudinal ventral, lateral and dorsal nerve cords, connected at intervals by transverse connectives. The CNS is better developed ventrally than dorsally or laterally, and has the strongest reactivity for all neuroactive substances examined. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts have been examined. Results implicate acetylcholine, FMRFamide-related peptides (FaRPs) and serotonin in sensory and motor function in this monogenean, although confirmatory physiological data are obviously required.

A new sensory organ in "primitive" molluscs (Polyplacophora Lepidopleurida), and its context in the nervous system of chitons

Introduction: Chitons (Polyplacophora) are molluscs considered to have a simple nervous system without cephalisation. The position of the class within Mollusca is the topic of extensive debate and neuroanatomical characters can provide new sources of phylogenetic data as well as insights into the fundamental biology of the organisms. We report a new discrete anterior sensory structure in chitons, occurring throughout Lepidopleurida, the order of living chitons that retains plesiomorphic characteristics.

Results: The novel "Schwabe organ" is clearly visible on living animals as a pair of streaks of brown or purplish pigment on the roof of the pallial cavity, lateral to or partly covered by the mouth lappets. We describe the histology and ultrastructure of the anterior nervous system, including the Schwabe organ, in two lepidopleuran chitons using light and electron microscopy. The oesophageal nerve ring is greatly enlarged and displays ganglionic structure, with the neuropil surrounded by neural somata. The Schwabe organ is innervated by the lateral nerve cord, and dense bundles of nerve fibres running through the Schwabe organ epithelium are frequently surrounded by the pigment granules which characterise the organ. Basal cells projecting to the epithelial surface and cells bearing a large number of ciliary structures may be indicative of sensory function. The Schwabe organ is present in all genera within Lepidopleurida (and absent throughout Chitonida) and represents a novel anatomical synapomorphy of the clade.

Conclusions: The Schwabe organ is a pigmented sensory organ, found on the ventral surface of deep-sea and shallow water chitons; although its anatomy is well understood, its function remains unknown. The anterior commissure of the chiton oesophagial nerve ring can be considered a brain. Our thorough review of the chiton central nervous system, and particularly the sensory organs of the pallial cavity, provides a context to interpret neuroanatomical homology and assess this new sense organ.

Consensus on Outcome Measures for Glaucoma Effectiveness Trials: Results From a Delphi and Nominal Group Technique Approaches

PURPOSE: Comparing the relative effectiveness of interventions across glaucoma trials can be problematic due to differences in definitions of outcomes. We sought to identify a key set of clinical outcomes and reach consensus on how best to measure them from the perspective of glaucoma experts.

METHODS: A 2-round electronic Delphi survey was conducted. Round 1 involved 25 items identified from a systematic review. Round 2 was developed based on information gathered in round 1. A 10-point Likert scale was used to quantify importance and consensus of outcomes (7 outcomes) and ways of measuring them (44 measures). Experts were identified through 2 glaucoma societies membership-the UK and Eire Glaucoma Society and the European Glaucoma Society. A Nominal Group Technique (NGT) followed the Delphi process. Results were analyzed using descriptive statistics.

RESULTS: A total of 65 participants completed round 1 out of 320; of whom 56 completed round 2 (86%). Agreement on the importance of outcomes was reached on 48/51 items (94%). Intraocular pressure (IOP), visual field (VF), safety, and anatomic outcomes were classified as highly important. Regarding methods of measurement of IOP, "mean follow-up IOP" using Goldmann applanation tonometry achieved the highest importance, whereas for evaluating VFs "global index mean deviation/defect (MD)" and "rate of VF progression" were the most important. Retinal nerve fiber layer (RNFL) thickness measured by optical coherence tomography (OCT) was identified as highly important. The NGT results reached consensus on "change of IOP (mean of 3 consecutive measurements taken at fixed time of day) from baseline," change of VF-MD values (3 reliable VFs at baseline and follow-up visit) from baseline, and change of RNFL thickness (2 good quality OCT images) from baseline.

CONCLUSIONS: Consensus was reached among glaucoma experts on how best to measure IOP, VF, and anatomic outcomes in glaucoma randomized controlled trials.

Diabetic retinopathy:morphometric analysis of basement membrane thickening of capillaries in different retinal layers within arterial and venous environments

AIMS: To assess quantitatively variations in the extent of capillary basement membrane (BM) thickening between different retinal layers and within arterial and venous environments during diabetes.

METHODS: One year after induction of experimental (streptozotocin) diabetes in rats, six diabetic animals together with six age-matched control animals were sacrificed and the retinas fixed for transmission electron microscopy (TEM). Blocks of retina straddling the major arteries and veins in the central retinal were dissected out, embedded in resin, and sectioned. Capillaries in close proximity to arteries or veins were designated as residing in either an arterial (AE) or a venous (VE) environment respectively, and the retinal layer in which each capillary was located was also noted. The thickness of the BM was then measured on an image analyser based two dimensional morphometric analysis system.

RESULTS: In both diabetics and controls the AE capillaries had consistently thicker BMs than the VE capillaries. The BMs of both AE and VE capillaries from diabetics were thicker than those of capillaries in the corresponding retinal layer from the normal rats (p < or = 0.005). Also, in normal AE and VE capillaries and diabetic AE capillaries the BM in the nerve fibre layer (NFL) was thicker than that in either the inner (IPL) or outer (OPL) plexiform layers (p < or = 0.001). However, in diabetic VE capillaries the BMs of capillaries in the NFL were thicker than those of capillaries in the IPL (p < or = 0.05) which, in turn, had thicker BMs than capillaries in the OPL (p < or = 0.005).

CONCLUSIONS: The variation in the extent of capillary BM thickening between different retinal layers within AE and VE environments may be related to differences in levels of oxygen tension and oxidative stress in the retina around arteries compared with that around veins.

Developing the clinical components of a complex intervention for a glaucoma screening trial:a mixed methods study

BACKGROUND: Glaucoma is a leading cause of avoidable blindness worldwide. Open angle glaucoma is the most common type of glaucoma. No randomised controlled trials have been conducted evaluating the effectiveness of glaucoma screening for reducing sight loss. It is unclear what the most appropriate intervention to be evaluated in any glaucoma screening trial would be. The purpose of this study was to develop the clinical components of an intervention for evaluation in a glaucoma (open angle) screening trial that would be feasible and acceptable in a UK eye-care service.

METHODS: A mixed-methods study, based on the Medical Research Council (MRC) framework for complex interventions, integrating qualitative (semi-structured interviews with 46 UK eye-care providers, policy makers and health service commissioners), and quantitative (economic modelling) methods. Interview data were synthesised and used to revise the screening interventions compared within an existing economic model.

RESULTS: The qualitative data indicated broad based support for a glaucoma screening trial to take place in primary care, using ophthalmic trained technical assistants supported by optometry input. The precise location should be tailored to local circumstances. There was variability in opinion around the choice of screening test and target population. Integrating the interview findings with cost-effectiveness criteria reduced 189 potential components to a two test intervention including either optic nerve photography or screening mode perimetry (a measure of visual field sensitivity) with or without tonometry (a measure of intraocular pressure). It would be more cost-effective, and thus acceptable in a policy context, to target screening for open angle glaucoma to those at highest risk but for both practicality and equity arguments the optimal strategy was screening a general population cohort beginning at age forty.

CONCLUSIONS: Interventions for screening for open angle glaucoma that would be feasible from a service delivery perspective were identified. Integration within an economic modelling framework explicitly highlighted the trade-off between cost-effectiveness, feasibility and equity. This study exemplifies the MRC recommendation to integrate qualitative and quantitative methods in developing complex interventions. The next step in the development pathway should encompass the views of service users.

Parasite neuropeptide biology:Seeding rational drug target selection?

The rationale for identifying drug targets within helminth neuromuscular signalling systems is based on the premise that adequate nerve and muscle function is essential for many of the key behavioural determinants of helminth parasitism, including sensory perception/host location, invasion, locomotion/orientation, attachment, feeding and reproduction. This premise is validated by the tendency of current anthelmintics to act on classical neurotransmitter-gated ion channels present on helminth nerve and/or muscle, yielding therapeutic endpoints associated with paralysis and/or death. Supplementary to classical neurotransmitters, helminth nervous systems are peptide-rich and encompass associated biosynthetic and signal transduction components - putative drug targets that remain to be exploited by anthelmintic chemotherapy. At this time, no neuropeptide system-targeting lead compounds have been reported, and given that our basic knowledge of neuropeptide biology in parasitic helminths remains inadequate, the short-term prospects for such drugs remain poor. Here, we review current knowledge of neuropeptide signalling in Nematoda and Platyhelminthes, and highlight a suite of 19 protein families that yield deleterious phenotypes in helminth reverse genetics screens. We suggest that orthologues of some of these peptidergic signalling components represent appealing therapeutic targets in parasitic helminths.

Paired associative transcranial alternating current stimulation increases the excitability of corticospinal projections in humans

Many types of non-invasive brain stimulation alter corticospinal excitability (CSE). Paired associative stimulation (PAS) has attracted particular attention as its effects ostensibly adhere to Hebbian principles of neural plasticity. In prototypical form, a single electrical stimulus is directed to a peripheral nerve in close temporal contiguity with transcranial magnetic stimulation delivered to the contralateral primary motor cortex (M1). Repeated pairing of the two discrete stimulus events (i.e. association) over an extended period either increases or decreases the excitability of corticospinal projections from M1, contingent on the interstimulus interval. We studied a novel form of associative stimulation, consisting of brief trains of peripheral afferent stimulation paired with short bursts of high frequency (≥80 Hz) transcranial alternating current stimulation (tACS) over contralateral M1. Elevations in the excitability of corticospinal projections to the forearm were observed for a range of tACS frequency (80, 140 and 250 Hz), current (1, 2 and 3 mA) and duration (500 and 1000 ms) parameters. The effects were at least as reliable as those brought about by PAS or transcranial direct current stimulation. When paired with tACS, muscle tendon vibration also induced elevations of CSE. No such changes were brought about by the tACS or peripheral afferent stimulation alone. In demonstrating that associative effects are expressed when the timing of the peripheral and cortical events is not precisely circumscribed, these findings suggest that multiple cellular pathways may contribute to a long term potentiation-type response. Their relative contributions will differ depending on the nature of the induction protocol that is used.