Article Category: Research Article
Publicado en línea: 01 jul 2015
Páginas: 29 - 38
DOI: https://doi.org/10.2478/gsr-2015-0003
© 2015 James Olumide Ashaolu et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
In response to chronic head-down positioning, laboratory rodents develop increased intracranial pressure and reduced caudal pressure (Papadopoulos and Delp, 2002). Hindlimb unloading rodent models have been used in ground-based studies to simulate the mechanical alterations that occur with exposure to the microgravity environment of spaceflight (Colleran et al., 2000; Papadopoulos and Delp, 2002; White, 2005). In order to advance the understanding of the mechanisms of arterial adaptation, there are advantages to studying animals with unique challenges imposed by long periods of head-down tilt and extreme inversion, as occurs in bats.
The degree of inversion maintained by bats at rest is greater than that achieved in hindlimb unloaded rodents. Bats normally rest and sleep inverted for about 12 hours each day. Other times, they fly long distances and feed on fruit in non-head-down postures. The specific objective of this work was to characterize the histological adaptation of the bat ascending and abdominal arterial walls during extended head-down posture.
Forty (40) presumably healthy fruit bats (
The bats were divided randomly into four groups. Group A bats were taken directly into the laboratory for immediate euthanasia. Groups B, C, and D underwent inversion for 8, 15, and 22 days, respectively. The bats were singly-housed in wooden cages. Within the cage, the bats hung inverted by their hook-shaped hindlimbs that gripped the wire gauze roof of the cage. This is the normal position during sleeping and resting. Throughout the period of the experiment, the roof of the cages to which all the bats hung their hindlimbs was unopened. The bats hanging was also unrestricted. Cage design did not permit bats crawling to the base of the cages. Also, food and water were positioned on a platform close to the roof. The bats aligned their bodies parallel to the Earth’s gravitational field, except when eating.
Body position of the bats was recorded twice a day (8 a.m. and 8 p.m.). Before readings were taken, ten minutes of waiting was observed after reaching the laboratory area where the bats were kept to limit bat reaction to human entrance. The bats adapted to human presence. Bats were recorded as being inverted when their trunk and head were tilted downward, and they were recorded as being non-inverted when their trunk and head were tilted upward or horizontally. It was observed that the bats were inverted in 98.2% of total observation and were non-inverted in only 1.8% of all observations.
The bats were weighed to calculate the dose of sodium pentobarbital (40 mg/kg) which was used for the euthanasia. When deeply anesthetized, a longitudinal incision was made through the mid-thoracic and mid-abdominal walls to obtain the aortae of the bats.
The ascending and abdominal aortae were carefully dissected and excised for histological processing as described by Culling (1974). The ascending aorta was excised at 1 cm away from its origin from the heart, and the abdominal aorta was excised at 6-7 cm distal from the heart. The tissues were fixed in 10% formal saline. The fixed tissues were dehydrated in a graded series of ethanol, cleared in xylene, and embedded in paraffin wax at 58°C in plastic cassettes. Cross sections were cut at 3 μm and affixed on clean slides. The serial sections were stained with Haematoxylin and Eosin (H&E, general structure), Orcein (elastic fibers), and Van Gieson (collagen fibers). Slides were examined by light microscopy and photomicrographs were taken with a high-definition digital camera, Leica ICC50 (Leica Microsystems, England), mounted on a microscope, Leica DM 750 (Leica Microsystems, England).
ScopePhoto 3.0.11(2007) software was used for histomorphometry at a final magnification of times 100. Every sixth serial section was analyzed to avoid replicate counting of measurement. For each parameter, measurements were taken from at least five sections from each animal. Values were averaged to generate means ± standard error of mean (SEM) for each group and used for statistical analysis. Vascular wall thickness was measured at four points (0°, 90°, 180°, and 270°) following the method of Lee, 1987. Tunica media thickness was measured by a line drawn from the luminal to the adventitial margins of the tunica media. Tunica adventitia was measured by a line drawn from the media-adventitia boundary of the vessel wall (Lee, 1987).
Values were expressed as means ± SEM and compared between the test groups and the control group using one-way Analysis of Variance, Scheffe’s post hoc test, and Least Significant Differences (SPSS statistical software, version 20). Multiple comparisons were also performed between all groups. Statistical significance was defined at a value of P<0.05.
Compared to control (A), the thickness of the tunica media of the ascending aorta is significantly greater after prolonged head-down posture with percentages of 35.0% (B), 49.8% (C), and 100.7% (D) (Figure1). The adventitial thickness is also higher after longer inversion in B (132.4%), C (159.5%), and D (365.8%) compared with control (A) (Figure 1). The ratio of adventitia to media thickness is larger (1.25) in group D than control (A) (0.51).
Figure 1.
The thicknesses of the media compared to control (A) are significantly smaller after inversion by 25.6% (B), 47.3% (C), and 49.4% (D) (Figure 2). The adventitial thicknesses also decreased in B (28.0%), C (25.6%), and D (29.3%), compared with control (A) (Figure 2). The ratio of adventitia to media thickness is larger (1.00) in group D than control (A) (0.72).
Figure 2.
The tunica media is thicker in B, C, and D, compared to control (A), and filled with smooth muscle cells (Figure 3). In control (A), collagen fibers appear as tightly interwoven strands throughout the width of the tunica media (Figure 4). In groups B, C, and D, collagen fibers fill the full width of the tunica media. Elastic fibers are present across the media (Figure 5). In control (A), elastin fibers are closely packed, whereas in groups B, C, and D, they are less densely packed and appear more tortuous and interwoven (Figure 5).
Figure 3.
Figure 4.
Figure 5.
The reduced thickness of the tunica media and adventitia is evident in H&E sections from groups B, C, and D compared to control (A) (Figure 6). There appears to be less collagen in the adventitia of groups B, C, and D compared to the robust collagen density in control (A) (Figure 7). Elastin fibers are not different in control and test groups (staining not shown).
Figure 6.
Figure 7.
Our study is the first to examine aortic and morphological adaptation in bat prolonged inversion. The aortic adaptations are region-specific involving tunica media and adventitia. There is markedly increased thickness of the tunica media of the ascending aorta and thinning of the media of the abdominal aorta. The observed aortic wall adaptation in the current study might have been potentiated by a shift of intravascular fluid from the caudal part of the bats to the cranial part and, consequently, exposed the ascending aorta to greater mechanical stress while the abdominal aorta was less stressed. The increased media smooth muscle adaptation in the ascending aorta of bats might produce enhanced myogenic responses (Heather et al., 2006). Gao et al. (2009) examined morphological adaptations associated with hindlimb unloading in rats. They observed that 28 days of head-down tilt induced mild hypertrophic changes in the common carotid artery of the abdominal aorta. Our findings in inverted bats are similar, but more striking than in the rat model (Tuday et al., 2007). Meanwhile, this may be due to greater angle of tilt (vertical to gravity vector) at which bats are inverted compared to rodents. The observed growth of the ascending aorta could lead to aortic regurgitation and could partly contribute to cardiac myocyte damage seen in the bat heart in prolonged inversion (Ashaolu and Ajao, 2014). The rapid hypertrophy of the ascending aorta wall may be an adaptive response to counteract increased cranial pressure. Vascular physiological assessment is required to check for predicted functional changes.
Judging from the histological sections, the collagen and elastin content within the ascending aorta increased in-step with the smooth muscle growth. This is important for maintaining strength and function of the arterial wall under presumed conditions of increased pressure. Increased collagen is consistent with that reported for the thoracic aorta in head-down tilted rodents (Tuday et al., 2009).
Opposite to the hypertrophic response in the ascending aorta, the abdominal aorta exhibits involution. It would be of interest to compare the physiological responses of ascending and abdominal regions of the aorta to increased luminal pressure. Reduced wall thickness may result in hyporesponsiveness of the aortic segment pressure. It has been demonstrated in inverted laboratory animals that decreased pressure existed within the abdominal aortic column (Heather et al., 2006), and most studies have established atrophic changes in mesenteric vessels (Heather et al., 2006). The present study has demonstrated a progressive reduction in the wall thickness and collagen content in the abdominal aorta as inversion days progressed. The decreased collagen component of the abdominal aorta implies reduced stiffness. Previously, Ashaolu (2009) hypothesized that bat abdominal vessels may serve as blood reservoir during inversion. The present observations reveal a dramatic adaptation of the aorta, and changes are region-specific. With respect to stress on captive bats, our findings reveal that the housing conditions can profoundly change vascular anatomy. At a more general level, the plasticity of the aorta uncovered in this research suggests that humans in microgravity with altered blood flow and pressure distributions from the Earth may experience vascular remodeling that puts them at risk for cardiovascular malfunction during return to gravity.
The head-down posture resulted in wall thickening of the ascending aorta and thinning of the descending portions of the aorta. The structural changes are considered to be dramatic and consistent with regions of higher and lower intra-arterial pressures. This study sheds light on the arterial adaptations that are associated with profound gravitational vector alterations in bats. Further studies will be required to discover what extent this adaptability of arteries exists in other species, including humans.