Effect of local shell conductance on the vascularisation of the chicken chorioallantoic membrane

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Abstract

The vascularisation of the chorioallantoic membrane (CAM) of avian embryos is influenced by environmental oxygen partial pressure (PO2) on a global level: incubation at high PO2 reduces the density of pre- and post-capillary vessels of the CAM and decelerates the thinning of the blood–gas barrier, and vice versa. This study investigates the effects of local PO2 on vascular development during the formative period of days ten to fifteen, by making half of the egg hypoxic and the other half hyperoxic. The densities of arterioles, venules and capillaries were reduced under the hypoxic side, compared to untreated eggs, but not significantly changed on the hyperoxic side. Harmonic mean thickness of the tissue barrier and total CAM blood volume were not affected by the treatments. Vascular development of the CAM was therefore only partly influenced by local PO2.

Introduction

Gas exchange of the avian embryo takes place mainly by diffusion (Wangensteen et al., 1970/71; Paganelli, 1980; Metcalfe et al., 1981; Rahn et al., 1987). The diffusion rate is proportional to the difference of the partial pressure of a gas between the two sides of a barrier and to the permeability of that barrier (conductance). Bird embryos exchange respiratory gases with the environment though a barrier that has several layers. These are commonly grouped into an outer barrier, formed by the shell and the outer shell membrane, and an inner barrier formed by the inner shell membrane, its limiting film and the tissue of the chorioallantoic membrane (CAM), the major respiratory organ of the avian embryo, (Wangensteen, 1972; Piiper et al., 1980; Wangensteen and Weibel, 1982; Seymour and Piiper, 1988). The major contributor to the diffusive resistance of the outer barrier is the eggshell. The outer shell membrane contributes only little resistance to the gas flow after it has dried (Wangensteen et al., 1970/71; Paganelli et al., 1978; Piiper et al., 1980; Wangensteen and Weibel, 1982). Similarly, the inner shell membrane has a low diffusion resistance after drying, except for its limiting film (Kayar et al., 1981; Tranter et al., 1983; Visschedijk et al., 1988; Ar and Girard, 1989). The tissue barrier may also have only a very low resistance after the maturation of the CAM (Wangensteen and Weibel, 1982), and the greatest part of the resistance of the inner barrier to oxygen transport has been attributed to the oxygen binding in the blood (Piiper et al., 1980; Wangensteen and Weibel, 1982). In the second half of incubation, the eggshell may thus provide the greatest resistance of the mechanical barrier for the gas flow to and from the avian embryo (Ar et al., 1974).

Shell conductance depends on the number and size of its pores (Ar and Rahn, 1985; Rahn et al., 1987). Pore density differs between different regions of the chicken egg (Rokitka and Rahn, 1987; Seymour and Visschedijk, 1988), and in accordance with this observation different gas compositions have been found under different parts of the egg (Sotherland et al., 1984; Booth et al., 1987), regional differences maintained by a low lateral diffusion in the shell membranes under the shell (Visschedijk et al., 1988).

On a global (whole-egg) level, the development of the CAM is adaptable and matches the requirements of the embryo. For example, the global density of pre- and post-capillary vessels in the CAM is increased or decreased in response to an altered gas environment (Dusseau and Hutchins, 1988; Strick et al., 1991). A global response is also found in the chorioallantoic capillaries. They form a system of interconnected vessels in the chorionic epithelium that is better described as a plexus than as a network (Burton and Palmer, 1989). From day 8 onwards, the capillaries move through the outer epithelium, until day 14, when only a very thin layer of attenuated cytoplasmatic processes remains between the blood and the gas space in the shell membranes (Fitze-Gschwind, 1973; Ausprunk et al., 1974). This minimises the diffusive distance for the exchange of respiratory gases and serves to optimise respiration during development (Fitze-Gschwind, 1973; Wangensteen and Weibel, 1982), and it becomes crucial during late incubation when oxygen levels in the egg are lowest. Burton and Palmer (1992) showed that this process of capillary invagination may be accelerated or decelerated in hypoxic or hyperoxic environment, respectively. Under normal incubation the density of the larger pre- and post-capillary vessels continues to increase until day 14 (Kurz et al., 1995), while proliferation of the capillary plexus is greatly reduced after day 10 of development (Ausprunk et al., 1974; DeFouw et al., 1989; Wilting and Christ, 1993, Wilting and Christ, 1996).

In order to achieve maximum efficiency of gas exchange, vascularisation and blood flow in the CAM should match shell conductance (gas pressures) not just on a global level, but also locally. Not to distribute blood flow according to local shell conductance would lead to a diffusion/perfusion mismatch that may cause functional hypoxia and possibly reduce respiration and growth. However, no such reduction was observed under conditions of artificially enhanced local differences in shell conductance (Wagner-Amos and Seymour, 2002). Some matching of local CAM perfusion and shell conductance had been previously observed by Seymour and Visschedijk (1988). The aim of our study was therefore to investigate whether the avian embryo is able to adapt the development of the CAM vascular system and its perfusion locally and therefore achieve a match of diffusion and perfusion under locally variable shell conductance. To this effect, local reduction of shell conductance was achieved by waxing half of the eggshell, while on the other side (hyperoxic side) an increase of shell conductance was simulated by incubation in a dry, hyperoxic environment (wax/oxygen treatment), and the adjustments of vascular density, blood volume, and microvasculature of the CAM were compared between control eggs and those that had been treated with local changes in shell conductance.

Section snippets

Egg incubation

Fresh fertile chicken eggs (crossbred) were obtained from a local breeder (Globe Derby Poultry). Immediately after delivery, they were cleaned, weighed to the nearest mg (Mettler AE 163 Dual Range Balance) and stored as described in Wagner-Amos and Seymour (2002).

Incubation was started when eggs were transferred to a desiccator for the measurement of shell conductance for 24 h as described in Wagner-Amos and Seymour (2002) based on the method by Ar et al. (1974) using Fick's equation of

Density of pre- and post-capillary vessels

From day 10, the density of pre- and post-capillary (PPC) vessels increased in control eggs (p=0.0052, χ2=12.754; Kruskal–Wallis test), reaching a maximum on day 14 (Fig. 1). The wax/oxygen treated eggs showed an increase from day 12 to day 15 on the hyperoxic side (p=0.0009, F2,22=9.7696, ANOVA), but not under the waxed side. Pre- and post-capillary densities under the wax side were always less than the hyperoxic side of the same eggs (p=0.059 day 12, p=0.050 day 14, p=0.006 day 15, paired

Vessel density and blood volume

The vascularisation of the CAM was influenced by low shell permeability. The density of pre- and post-capillary blood vessels (PPC) under the wax side of experimental eggs was reduced from days 12 to 14 by 15.0–32.7% in comparison to the hyperoxic side (Fig. 1). Further, capillary surface area under the waxed shell was lower than on the hyperoxic side by 35.3% on day 12 to 59.2% on day 14 (Fig. 5). This indicates that angiogenesis was reacting negatively to the strongly hypoxic environment at

Acknowledgements

This study was supported by the Australian Research Council and the University of Adelaide. We thank Sue Runciman for advice on stereological techniques and analysis.

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