Layne L. Wright

The immunocytochemical detection of amino-acid neurotransmitters in paraformaldehyde-fixed tissues

In this study, we show that specific antibodies can be raised against paraformaldehyde conjugates of amino acids, including the neurotransmitters glycine, gamma-amino-butyric acid and glutamate, and a non-neuroactive amino acid, glutamine. These antibodies against paraformaldehyde conjugates specifically detect the above amino acids in paraformaldehyde-fixed tissues. The penetration of antibodies into paraformaldehyde-fixed tissues is much superior to the penetration of antibodies into glutaraldehyde-fixed tissues; hence good labeling can be observed through the depth of the tissues. Unlike glutaraldehyde, fixation with paraformaldehyde does not give rise to high levels of tissue autofluorescence and, thus, these antibodies are very effective for immunofluorescence studies. Furthermore we suggest that the ability of these antibodies to detect amino acids in paraformaldehyde-fixed tissues will permit their use in situations where it is necessary to detect other other fixation-sensitive antigens, such as neurotransmitter receptors and transporters.

The type 1 polyaxonal amacrine cells of the rabbit retina: a tracer-coupling study.

The type 1 polyaxonal (PA1) cell is a distinct type of axon-bearing amacrine cell whose soma commonly occupies an interstitial position in the inner plexiform layer; the proximal branches of the sparse dendritic tree produce 1-4 axon-like processes, which form an extensive axonal arbor that is concentric with the smaller dendritic tree (Dacey, 1989; Famiglietti, 1992a,b). In this study, intracellular injections of Neurobiotin have revealed the complete dendritic and axonal morphology of the PA1 cells in the rabbit retina, as well as labeling the local array of PA1 cells through homologous tracer coupling. The dendritic-field area of the PA1 cells increased from a minimum of 0.15 mm2 (0.44-mm equivalent diameter) on the visual streak to a maximum of 0.67 mm2 (0.92-mm diameter) in the far periphery; the axonal-field area also showed a 3-fold variation across the retina, ranging from 3.1 mm2 (2.0-mm diameter) to 10.2 mm2 (3.6-mm diameter). The increase in dendritic- and axonal-field size was accompanied by a reduction in cell density, from 60 cells/mm2 in the visual streak to 20 cells/mm2 in the far periphery, so that the PA1 cells showed a 12 times overlap of their dendritic fields across the retina and a 200-300 times overlap of their axonal fields. Consequently, the axonal plexus was much denser than the dendritic plexus, with each square millimeter of retina containing approximately 100 mm of dendrites and approximately 1000 mm of axonal processes. The strong homologous tracer coupling revealed that approximately 45% of the PA1 somata were located in the inner nuclear layer, approximately 50% in the inner plexiform layer, and approximately 5% in the ganglion cell layer. In addition, the Neurobiotin-injected PA1 cells sometimes showed clear heterologous tracer coupling to a regular array of small ganglion cells, which were present at half the density of the PA1 cells. The PA1 cells were also shown to contain elevated levels of gamma-aminobutyric acid (GABA), like other axon-bearing amacrine cells.

Expression of lectin binding in the superficial dorsal horn of the rat spinal cord during pre- and postnatal development

The plant lectin Bandeiraea simplicifolia I-B4 binds to the soma and central terminals of a subpopulation of unmyelinated primary sensory neurones in the adult rat. The binding site of this lectin is thought to be the terminal alpha-D-galactose residue of a membrane associated glycoconjugate which may be involved in the development of specific connections between small diameter primary sensory neurones and second order neurones in the superficial dorsal horn of the spinal cord. To begin to investigate this possibility we have examined the development of lectin binding in the dorsal horn of pre- and postnatal rats. Lectin binding first appeared on axon profiles in the superficial dorsal horn of the spinal cord at embryonic days 18/19. Previous studies in the rat have revealed that the central processes of small diameter primary sensory neurones enter the dorsal horn at embryonic days 18/19. Our findings suggest that the glycoconjugate to which this lectin binds, is expressed by the central processes of small diameter primary sensory neurones as they grow into the spinal cord. It is therefore possible that this glycoconjugate is involved in the development of topographically ordered neural connections within the dorsal horn of the spinal cord.

Effects of glucocorticoid exposure on growth and structural maturation of the heart of the preterm piglet.

Inadequate maintenance of systemic blood flow in neonates following preterm birth is associated with increased morbidity and mortality, and may be due in part to structural immaturity of the myocardium. Maternal glucocorticoid administration is associated with improved cardiovascular function, and possibly promotes structural maturation of the myocardium. This study assessed the structural maturity of the myocardium in male and female preterm and term piglets, and preterm piglets exposed to a regimen of maternal glucocorticoids as used clinically. In preterm, term and glucocorticoid exposed preterm piglets cardiomyocyte maturity was examined by measuring the proportion of binucleated myocytes and the volumes of single living ventricular cardiomyocytes with fluorescence microscopy. Ventricular apoptosis and proliferation were measured by immunohistochemistry. Preterm piglet hearts had fewer binucleated myocytes, smaller myocytes, and more proliferative and fewer apoptotic nuclei than term hearts. Maternal glucocorticoid treatment resulted in increased binucleation with no increase in myocyte volume, and levels of proliferation and apoptosis that were more similar to the term heart. Atrial weights were increased and in female piglets there was an increase in the ratio of left to right ventricular weight. The observed changes in atrial mass and myocyte structural maturation correlated with changes in cardiac function of isolated hearts of littermates. In conclusion, the association between increased myocardial maturation following glucocorticoid exposure, improved cardiac function in littermates, and clinical improvement in human neonatal cardiac function exposed to antenatal glucocorticoids, suggests that glucocorticoid exposure contributes to improved cardiovascular function in preterm infants by promoting myocardial structural maturity.

Endogenous angiotensins and catecholamines do not reduce skin blood flow or prevent hypotension in preterm piglets

Endocrine control of cardiovascular function is probably immature in the preterm infant; thus, it may contribute to the relative ineffectiveness of current adrenergic treatments for preterm cardiovascular compromise. This study aimed to determine the cardiovascular and hormonal responses to stress in the preterm piglet. Piglets were delivered by cesarean section either preterm (97 of 115 days) or at term (113 days). An additional group of preterm piglets received maternal glucocorticoids as used clinically. Piglets were sedated and underwent hypoxia (4% FiO2 for 20 min) to stimulate a cardiovascular response. Arterial blood pressure, skin blood flow, heart rate and plasma levels of epinephrine, norepinephrine, angiotensin II (Ang II), angiotensin‐(1–7) (Ang‐(1‐7)), and cortisol were measured. Term piglets responded to hypoxia with vasoconstriction; preterm piglets had a lesser response. Preterm piglets had lower blood pressures throughout, with a delayed blood pressure response to the hypoxic stress compared with term piglets. This immature response occurred despite similar high levels of circulating catecholamines, and higher levels of Ang II compared with term animals. Prenatal exposure to glucocorticoids increased the ratio of Ang‐(1‐7):Ang II. Preterm piglets, in contrast to term piglets, had no increase in cortisol levels in response to hypoxia. Preterm piglets have immature physiological responses to a hypoxic stress but no deficit of circulating catecholamines. Reduced vasoconstriction in preterm piglets could result from vasodilator actions of Ang II. In glucocorticoid exposed preterm piglets, further inhibition of vasoconstriction may occur because of an increased conversion of Ang II to Ang‐(1‐7).

A Pig Model of the Preterm Neonate: Anthropometric and Physiological Characteristics

Background and Aims Large animal models are an essential research tool to investigate the physiology of the preterm infant, which remains poorly understood. We aim to describe the pig model of the preterm neonate in terms of growth, maturation and requirement for intensive care over a range of gestational ages and determine the effects of maternal glucocorticoid exposure and sex. Methods Twenty-nine litters of piglets (N=305) were delivered by C-section at 91d, 94d, 97d, 100d, 104d and 113d (term 115d). Some litters received maternal betamethasone treatment (0.19mg/kg body wt; IM) at 48h and 24h prior to delivery. At 97d piglets were resuscitated, surfactant administered, and piglets were ventilated, sedated and monitored for 6–8h post-birth using standard NICU techniques. Results At 91d, piglets were half the weight of term animals, had fused eyelids, very thin skin, no hair, and survived a maximum of 3h due to difficulties with ventilation. At 97d piglets were able to be maintained for at least 6–8h but physiology was unstable for 1–2h. Piglets 100d and older breathed spontaneously. Only near term piglets were able to maintain body temperatures. Males were heavier than females at 113d gestation (p=0.021). Exposure to maternal glucocorticoids resulted in larger females and influenced brain:body wt. Conclusions The piglet provides a useful model of preterm neonatal physiology as very preterm piglets can be survived under standard intensive care conditions. The large litters allow for parallel experiments or the use of littermates as controls.

1131 Cardiovascular and Hormonal Responses to Hypoxic Stress in the Preterm Piglet

Background and Aims Immature control of the cardiovascular system may be a contributing factor to poor cardiovascular function and associated increases in mortality and morbidity in preterm infants. This study aimed to assess cardiovascular and hormonal responses to a mild hypoxic stress in newborn piglets. Methods Piglets were delivered by C-section at 97 and 113 days of gestation (term≈115d). An additional preterm group was exposed to maternal glucocorticoid treatment. Changes in mean arterial blood pressure (MAP), skin blood flow and plasma concentrations of adrenalin, noradrenalin, angiotensin II and cortisol were measured in response to acute hypoxia (4% O2 for 20min). Results Preterm piglets were less likely to exhibit a mature cardiovascular compensatory response to hypoxia (increased MAP and reduced skin blood flow) than term piglets. Plasma adrenalin and noradrenalin concentrations at the end of hypoxia were increased in all groups and were 2–3 fold higher in preterm pigs than in term pigs (P<0.05). Plasma cortisol levels were increased at the end of hypoxia in term piglets (P<0.05) but not in preterm or glucocorticoid exposed preterm piglets. Angiotensin II levels were reduced in glucocorticoid exposed preterm piglets compared to untreated preterm piglets (P<0.05). Conclusions The reduced cardiovascular compensatory response to hypoxia in preterm pigs is not the result of reduced plasma levels of catecholamines during hypoxia. Low levels of angiotensin II in glucocorticoid exposed preterm piglets may contribute to poor control of skin blood flow during hypoxia.