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First published online February 12, 2007
Journal of Experimental Biology 210, 815-824 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.001867
Morphometry of retinal vasculature in Antarctic fishes is dependent upon the level of hemoglobin in circulation
1 School of Marine Sciences, University of Maine, 5751 Murray Hall, Orono,
ME 04469-5751, USA
2 Department of Biology, University of Washington, Box 351800, Seattle, WA
98195-1800, USA
3 Department of Biomedical Sciences, Ohio University, Athens, OH 45701-2979,
USA
* Author for correspondence (e-mail: bsidell{at}maine.edu)
Accepted 3 January 2007
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1.5 times greater than vessel diameters of +Hb species (–Hb,
0.193±0.006 mm; +Hb, 0.125±0.005 mm). Vascular density index
(VDI), a stereological index that is affected by both vessel number and
length, is greatest in –Hb C. aceratus (3.51±0.20) and
lowest in +Hb N. coriiceps (1.58±0.14). Among four +Hb
species, there is a direct relationship between red blood cell content and
retinal vasculature. Hematocrit (Hct) is inversely correlated to vascular
density (r2=0.934) and positively correlated to
intervessel distance (r2= 0.898) over a >2.3-fold range
of Hct. These results indicate that anatomical capacity to supply blood to the
retina increases to compensate for decreases in oxygen-carrying capacity of
the blood.
Key words: Antarctic fish, hemoglobin, retina, vascular density, icefish, notothenioid, hematocrit, nitric oxide, circulation, morphometry
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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). It is the
coldest, densest, and most thermally stable marine habitat on the planet.
Continental shelf waters continuously hover near or below 0°C and vary
little seasonally or as a function of water depth
(Knox, 1970;
Lewis and Perkin, 1985).
Although presenting a number of physiological challenges, cold temperatures do
have a benefit. Because gaseous solubility is inversely proportional to
temperature, oxygen content in Antarctic waters is extremely high. These
conditions preclude marine organisms of the Southern Ocean from experiencing
environmental hypoxia under normal circumstances
(Littlepage, 1965).
Despite the harsh environmental conditions, one group of fishes has
flourished in the Southern Ocean. Species of the perciform suborder
Notothenioidei dominate the fish fauna of waters south of the Antarctic Polar
Front. Notothenioids encompass 8 families, 44 genera, and 129 species and
account for approximately 45% of fish species and 90% of fish biomass in the
region (Eastman, 1993;
Eastman, 2005). Notothenioids
radiated to dominate this marine system from an ancestral stock that survived
a dramatic crash in fish species diversity of the Southern Ocean that occurred
sometime within the last ca. 30 million years. Modern notothenioids occupy a
variety of habitat niches; some species are benthic or epibenthic, while
others are pelagic, semipelagic or cryopelagic
(Gon and Heemstra, 1990).
Activity levels vary among species from sedentary to active.
Similar to all other vertebrate groups, seven of the eight families of
fishes within the suborder Notothenioidei have blood with
hemoglobin-containing circulating erythrocytes. Hematocrits characteristic of
these red-blooded species overlap values typical of temperate zone fishes,
although absolute hemoglobin concentrations are generally lower than those of
temperate zone species because of lower mean corpuscular hemoglobin
concentrations (Egginton,
1996). Among red-blooded notothenioid fishes, behavioral
activities are correlated with concentrations of circulating hemoglobin (Hb)
and number of erythrocytes, which are higher in more active species
(Wells et al., 1980).
A single family of notothenioids, the channichthyid icefishes, are the only
known vertebrate animals to lack Hb completely as adults
(Ruud, 1954). In the absence
of Hb, oxygen is carried strictly in physical solution in icefish blood. This
results in an oxygen-carrying capacity in icefishes that is <10% of that
seen in closely related red-blooded species
(Holeton, 1970). Modifications
of the cardiovascular system of icefishes compensate for the complete absence
of oxygen-binding Hb. Icefishes possess large-bore capillaries and have blood
volumes that are 2–4 times larger than those of red-blooded fishes
(Fitch et al., 1984;
Hemmingsen and Douglas, 1970).
Additionally, hearts are larger in icefishes than in similar-sized red-blooded
fishes, resulting in mass-specific cardiac outputs that are several-fold
greater than those of red-blooded species
(Hemmingsen et al., 1972;
Holeton, 1970). Integration of
these features allows channichthyids to circulate large blood volumes at
relatively high flow rates. This is achieved at low arterial blood pressures
due to decreased systemic resistance to flow. Ultimately, the combination of
high-throughput cardiovascular systems, waters of high oxygen content, and low
absolute metabolic rates enables this group of fishes to deliver sufficient
oxygen to their tissues (Hemmingsen,
1991).
Hemmingsen and Douglas (Hemmingsen and
Douglas, 1970) first recognized that "The large blood
volume of C. aceratus may be a manifestation of an increased
vascularization." In retrospect, this conclusion is somewhat
axiomatic, given that the additional blood volume must be accommodated
somewhere within the vascular tree. Vasculature associated with the retina of
the eye provides an excellent example and is exceptionally dense in Hb-lacking
icefishes compared to Hb-expressing notothenioid species
(Eastman, 1988;
Eastman and Lannoo, 2004;
Sidell and O'Brien, 2006). The
vasculature of the teleost retina, especially in the case of channichthyids,
differs in a number of respects from that of mammals and other fishes. Unlike
mammals, most teleost retinae are avascular
(Chase, 1982) and lack retinal
arteries and veins within the substance of the retina. Furthermore, the
retinae of fishes are generally thick, with diffusion distances as much as six
times longer than in primates (Copeland,
1980). In order to satisfy the retinal requirement for oxygen, the
teleostean eye has a dual blood supply from an ophthalmic artery derived from
the pseudobranch and an optic artery from the internal carotid
(Nicol, 1989). There are also
three vascular structures not present in mammals: the choroid rete mirabile,
the lentiform body (also a rete), and the falciform process
(Walls, 1942;
Nicol, 1989). Although present
in phyletically basal notothenioids
(Eastman and Lannoo, in
press), channichthyids have lost the latter three structures and
the ophthalmic artery may be vestigal or lost as well
(Eastman and Lannoo, 2004).
Thus the channichthyid retina is supplied by an optic artery that branches at
the optic disk into an extensive series of hyaloid arteries at the
vitreoretinal interface (Fig.
1A). These arteries are thin-walled
(Eastman and Lannoo, 2004) and
serve as large bore arterial capillaries. Veins do not accompany the hyaloid
arteries in teleosts that have lost the ocular vascular structures mentioned
above (Copeland, 1980;
Nicol, 1989). This has been
documented in histological studies of the retinae of various notothenioids
(Eastman, 1988;
Eastman and Lannoo, 2003),
including channichthyids (Eastman and
Lannoo, 2004). The hyaloid arteries drain to an annular vein
located in the vicinity of the ora serrata at the retinal periphery
(Fig. 3B).
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Retinae of notothenioid fishes are highly aerobically poised in metabolism
(Waser and Heisler, 2004) and,
like brain (Kawall et al.,
2002), retinal oxygen demand should be relatively independent of
locomotory activity level in these species. Given that oxygen-carrying
capacity of blood can vary considerably, both among the red-blooded (+Hb)
notothenioids and certainly between those families and the hemoglobinless
(–Hb) icefishes, it seems reasonable to expect that anatomical and/or
physiological compensations must ensure adequate oxygenation of the tissue
among these fishes. The known physiological compensations of the
cardiovascular system in –Hb species are relatively well characterized,
as outlined above. To investigate possible anatomical compensations, we
undertook the present study with objectives to: (1) quantify morphometric
parameters of retinal vasculature in several species of both +Hb and –Hb
Antarctic notothenioids and (2) assess the relationship between vascular
patterns and hemoglobin content of blood. Our results show a profound
correlation between vascular patterns and hemoglobin content and further
suggest an underlying mechanism for regulating this relationship.
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Tissue preparation
To visualize vascular features, blood vessels of fishes were filled with
MicrofilTM (Flow Tech, Inc., Carver, MA, USA), a liquid silicon rubber
compound. Animals were first anesthetized with MS-222 in seawater (1:7500 w/v;
3-aminobenzoic acid ethyl ester; Sigma-Aldrich, St Louis, MO, USA) for a
period of 10–15 min. After initial anesthetization, a blood sample
(approximately 1 ml) was taken by caudal venipuncture from each red-blooded
individual for determination of hematocrit (Hct). Next, concentrated
heparinized notothenioid Ringer solution (2500 U ml–1 heparin
added to 260 mmol l–1 NaCl, 2.5 mmol l–1
MgCl2·6H2O, 5.0 mmol l–1 KCl,
2.5 mmol l–1 NaHCO3, 5.0 mmol l–1
NaH2PO4·H2O, pH 8.0) was injected into
the caudal vein at a dose of 2.5 ml kg–1 and the animal
was promptly returned to a holding tank for an additional 10 min. Fishes were
then placed ventral side up on an iced surgical platform and a series of
incisions were made to open the pericardial cavity. The atrium and ventricle
were removed, leaving the bulbus arteriosus intact in situ. The
bulbus arteriosus was cannulated with PE-tubing (90, 100, 160 or 190) and
ligated with surgical silk. The cannula was then connected to tubing leading
to a syringe loaded in a Model 100 syringe pump (KD Scientific Inc.,
Holliston, MA, USA). Approximately 15–30 ml of ice-cold heparinized
notothenioid Ringer solution (760 U ml–1 heparin added to 260
mmol l–1 NaCl, 2.5 mmol l–1
MgCl2·6 H2O, 5.0 mmol l–1 KCl,
2.5 mmol l–1 NaHCO3, 5.0 mmol l–1
NaH2PO4·H2O, pH 8.0) was perfused
throughout the entire vascular system at a flow rate of 
46.8 ml
h–1. Specimens were then filled with 9–27 ml of
ice-cold yellow MicrofilTM using the same apparatus at a flow rate of
34.2 ml h–1 until venous return of the compound to the
pericardial cavity was observed. While allowing the MicrofilTM to
polymerize, specimens were maintained on ice for about 1 h, followed by a
1-week fixation in 10% formalin and subsequent storage in 70% ethanol.
Fixed specimens were transported back to our home laboratory at the University of Maine where eyes were excised from each specimen as part of an intact, rectangular block of tissue, approximately 3.5 cmx5.0 cm in dimension. To visualize the hyaloid arteries at the vitreoretinal interface, the cornea, iris and lens were removed anterior to the margin of the ora serrata and the vitreous body was carefully extracted from the vitreous chamber.
Photography
Digital images were taken using a Nikon D70 camera (Nikon Inc., Melville,
NY, USA) fitted with a 60 mm AF Micro Nikkor lens (Nikon Inc., Melville, NY,
USA) and mounted on a stand approximately 27–28 cm above the plane of
the vessels. Images were shot in aperture mode (F51) to compensate for the
large depth of field of the eye. During the photographic procedure, eyes were
secured in a shallow Petri dish and covered with 70% ethanol. Several
photographs were taken of each eye and the best image of the series was chosen
for further processing.
Image processing
Original digital images were processed using ImageJ (Version 1.32; NIH,
Bethesda, MD, USA). Images were cropped by centering an oval region of
interest tool (ROI) over the optic disc and extending the ROI outward until
point of contact with the retractor lentis, forming a reference field
containing the majority (>90%) of the hyaloid arteries present at the
vitreoretinal interface (Fig.
1A,B). This procedure eliminated peripheral sections of the image,
which would be most subject to distortion due to curvature, and also ensured
that equivalent relative reference fields of eyes were sampled despite both
intrapecific and interspecific differences in eye size. Next, an RGB color
split was performed to isolate the yellow vessels. An edge finder, FJ
Laplacian plugin with a derivative smoothing scale of 3, was used on the
yellow channel image to maximize detection of the vessels from the background.
The yellow channel was inverted and contrast and brightness enhanced by
histogram equalization. We removed the remaining image noise by subtracting
the original blue channel values from this enhanced edge image, and again
enhanced brightness and contrast through histogram equalization. The resultant
image was then converted to binary via the threshold function
(Fig. 1C). For a few of the
images, there were discrete areas where vessels did not fill with
MicrofilTM. These regions were cropped from the reference field. As a
processing control, vessel diameters from the binary image were compared to
vessel diameters of the original image. Differences between the two never
exceeded 5%.
Procedure for inadequately filled specimens
A successful vascular fill was considered to be one where 
90% of the
hyaloid arteries on the vitrad surface of the retina were filled with
MicrofilTM. T. hansoni and N. coriiceps did not meet
this criterion; therefore an alternative method was devised to demonstrate the
ocular vasculature of these species. Tissues were grossly stained with a
solution of 1 part of 1% aqueous aniline blue in 10 parts of saturated aqueous
picric acid for 1–2 min. After rinsing with distilled water, each
eye was dissected further and returned to 70% ethanol. Blood vessels were then
discernable and further removal of the vitreous body was possible without
destruction of previously obscured vessels. For most of the specimens,
staining and subsequent dissection had to be repeated several times before the
tissue was satisfactory for photography.
Digital images of the stained eyes were taken as previously described. Using Adobe Photoshop (Version 7.0; Adobe Systems, Inc., San Jose, CA, USA) each image was converted to grayscale and contrast was adjusted to give maximal differentiation between the vessels and background. A transparent layer was superimposed on top of the grayscale image and blood vessels were traced using a digitizing tablet (DrawingBoard III; GTCO CalComp Inc., Columbia, MD, USA), while simultaneously verifying the actual vessels under a dissecting microscope. The brightness and contrast of the digitized drawing were adjusted so that the grayscale background layer disappeared (turned white) and the vessel layer (already black) remained, thus completing the conversion of the image to binary.
Image analysis
Morphometric measurements of retinal vascularity were obtained using an
automated macro developed in Matlab and the Matlab Image Processing Toolbox
(Matlab Version 7.1; Matlab Image Processing Toolbox Version 5.02; The
Mathworks, Inc., Natick, MA, USA). The design of the macro was based on
previous studies analyzing angiogenic response
(Maas et al., 1999;
Strick et al., 1991). A
concentric circle overlay was superimposed on each computerized binary image
with the first circle centered directly over the optic disc
(Fig. 1D). The diameter of the
innermost circle was 1.0 mm and spacing between successive circles was fixed
at 0.5 mm. Vascular density index (VDI), a stereological index that is
affected by both blood vessel length and number, was computed by dividing the
number of vessel–grid intersections by the total circumference of
circles within the reference field. VDI was first calculated for the entire
reference field of the vitreoretinal interface. Each image was entered into
Matlab twice and the average of the counts was recorded as the mean VDI for
that specimen. The difference between the two counts was used to assess
intra-observer variability. The reference field was then divided into four
quadrants (QI-ventrotemporal, QII-dorsotemporal, QIII-dorsonasal,
QIV-ventronasal), with the optic disc as the point of origin, to analyze VDI
as a function of location within the fundus.
In addition to VDI, several other morphometric measurements were obtained
using the Matlab macro. Individual vessel diameters, measured to the outside
vessel wall, were recorded at each vessel–grid intersection, averaged
together and reported as a mean for the entire eye. The intervessel distance
(IVD) for each gridline was calculated by dividing the circumference of the
circle by the number of intersecting vessels. Once again, calculations were
averaged for the entire vitreoretinal interface and IVD was reported as a
mean. Length density, the length of vessels per unit area, was derived using
the principle of Buffon by multiplying VDI by 
/2
(Weibel, 1979). Data were
collected for one other morphometric parameter; ImageJ was used to measure
fractional image area (FIA), the percent of reference field covered by blood
vessels. FIAs were calculated for entire eyes and quadrants.
Statistical analyses
Comparisons among the six species for each morphometric measurement were
performed in SigmaStat (Version 3.1; Systat Software, Inc., Point Richmond,
CA, USA) using a one-way ANOVA with a post-hoc
Student–Newman–Keuls test for multiple pairwise comparisons of the
means. The alpha value was set at P0.05. Linear least-squares
regression was used to analyze the relationship between Hcts and the
following: vascular densities, intervessel distances, specimen total lengths
and specimen body masses. Additionally, intra-observer variability associated
with operation of the Matlab macro was assessed. Variability was calculated by
dividing the standard deviation of the paired differences between replicates
by the overall mean VDI.
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General vascular patterns and morphometries
The overall vascular pattern in retinas of the six species examined appears
to be influenced by erythrocyte content of their blood. The greatest vascular
densities and most complex branching patterns are seen in hemoglobinless
(–Hb) icefishes. C. aceratus and C. gunnari display
branching patterns where four or five main arteries exit the optic disc and
divide into an anastomosing array of closely spaced parallel channels on the
fundus of the eye (Fig. 2A,B).
Both bathydraconid species showed a radial branching pattern of hyaloid
arteries with the vascular density of G. acuticeps greater than that
of P. charcoti. The latter species shows a density of arteries that
is clearly lower than those of icefishes
(Fig. 3A,B). Further reductions
in densities of radially arranged arteries in the retina are observed in the
red-blooded nototheniid species, T. hansoni and N. coriiceps
(Fig. 4A,B). N.
coriiceps shows the most sparsely distributed vascular array of any
species examined.
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1.5-fold greater than those
of +Hb bathydraconid species (vessel diameters of nototheniid species could
not be estimated for technical reasons – see Materials and methods). The
combination of these features, i.e. greatest length densities and vessel
diameters, in icefishes results in significantly greater FIAs (percent of
reference area covered by blood vessels) than seen in +Hb bathydraconids. In
combination, these observations indicate that species of fish that lack Hb
have larger blood vessel surface areas available for gas exchange and possess
vascular capacities capable of accommodating larger blood volumes than +Hb
fishes.
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In all species examined, FIA also varies as a function of location on the fundus of the eye. Ventrotemporal and ventronasal regions of the eye generally display the greatest FIA, indicating maximal blood vessel surface area in the region of the eye ventral to the optic disc (data not shown).
How close is the relationship between hematocrit and vascularity?
Two anatomical characteristics of the vasculature are particularly closely
related to capacity for supplying oxygen to tissue, vascular density index
(VDI) and intervessel distance (IVD). As its name implies, VDI is a
stereological index of vascularity within a given reference area. IVD is the
average path length between neighboring vessels and is thus equal to 2x
the shortest pathway for gaseous diffusion between vessels and underlying
retinal tissue. Assuming relatively similar rates of perfusion, one might
anticipate that reductions in oxygen-carrying capacity of blood might be met
with compensatory increases in VDI and decreases in IVD. To test this, we
examined the correlation of Hct with each of these parameters within the
red-blooded notothenioids examined in this study.
VDI shows a striking inverse correlation with Hct among the red-blooded species (r2=0.934; red symbols in Fig. 5A). Not surprisingly, hemoglobinless species that have the lowest blood oxygen-carrying capacity of any of the fishes studied also show the densest vasculature (Table 2). Because the hemoglobinless icefishes are known to have substantially higher cardiac outputs than their red-blooded relatives, however, they are apparently able to adequately oxygenate their retinal tissue at a lower vascular density than would be predicted by the relationship among the +Hb species (blue symbols, Fig. 5A). The icefish species were not incorporated in our regression analysis because of this marked difference in blood flow from red-blooded species. Examination of the relationship between Hct and IVD further illustrates the validity of this decision.
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An equally compelling correlation, in this case positive, exists between Hct and IVD among the red-blooded notothenioids (red symbols in Fig. 5B). As the oxygen delivery capacity per unit volume of blood increases, hyaloid arteries are spaced farther apart. Here, once again, the hemoglobinless icefishes that have the lowest blood oxygen-carrying capacities also show the closest spacing of arteries, but not to the point that would be extrapolated from the relationship among red-blooded species (Fig. 5B). If extrapolated from the relationship between Hct and IVD among red-blooded notothenioids, IVD for icefishes that are completely devoid of Hb would approach 0 mm, i.e. a complete vascular blanket of the visual surface. As with VDI above, the magnitude of anatomical compensation required in IVD is apparently diminished by marked greater perfusion of tissue in the –Hb icefishes.
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).
Because of this aerobic character, maintenance of adequate oxygen tensions is
likely to be an acute challenge in eyes of many Antarctic notothenioid fishes,
particularly for species of this group in which hemoglobin is expressed at low
levels or is completely absent. Previous reports have indicated that dramatic
differences exist in geometries of ocular blood vessels when comparing
hemoglobinless and red-blooded Antarctic fishes
(Eastman, 1988;
Eastman and Lannoo, 2004).
Those studies qualitatively described substantially higher vascular densities
on the vitrad surface of the retina in –Hb species than in +Hb
notothenioids but, with the exception of measuring vessel diameters, did not
quantify any other morphometric parameter of retinal vascular anatomy. Our
measurements provide a more thorough quantitative description of morphometries
in retinal vasculatures of several notothenioid fishes, corroborating and
extending the earlier qualitative accounts.
To quantify vascular parameters in eyes of notothenioid fishes, we
initially either filled the vasculature with a silicone rubber compound or
used a gross staining technique and then subsequently employed digital image
analysis. In recent years, digital methods have increasingly been employed to
quantify biological structures (Abrams et
al., 1994; Rieder et al.,
1995). Automated image analysis decreases processing time and
yields highly reproducible results. The software macro that we developed for
the present study resulted in intra-observer variation of <1%, a
substantial improvement over previous measurement variability with automated
systems (Maas et al., 1999;
Voss et al., 1984). Results of
our analyses demonstrate that the extent of retinal vascular proliferation is
directly related to the amount of Hb-containing erythrocytes in the
circulation.
Hemoglobinless icefishes display the most elaborate retinal vasculature
C. aceratus and C. gunnari, –Hb icefishes, display
a distinctive vascular branching pattern with 4–5 large hyaloid arteries
originating at the optic disc and anastomosing into closely spaced parallel
vessels. This pattern ultimately results in the highest density of blood
vessels among all the notothenioids examined. In bathydraconid species (modest
Hct levels), the pattern shifts to a more radial distribution, with 4–5
main arteries originating at the optic disc and branching dichotomously;
vascular densities also are lower (see below). The species with the highest
red cell content examined, N. coriiceps, displays a clear radial
distribution of very widely separated arteries.
Antarctic notothenioids display a more extensive and homogeneous pattern of
hyaloid arteries compared to the majority of teleosts studied to date
(Anctil, 1968;
Eastman, 1988;
Hanyu, 1962). Eastman
(Eastman, 1988) described the
notothenioid hyaloid pattern as being radially asymmetrical, with slightly
greater densities on the ventral and nasal fields. He observed no marked
differences in vessel distribution between central and peripheral regions of
the retina. This is unlike the unusual pattern seen in the surface-living
cyprinodontid, Fundulus grandis, where a highly vascularized area
centralis is observed ventral to the optic disc
(Copeland, 1976). Vascular
geometries of notothenioids that we examined generally conform to the above
characterization by Eastman, with greater densities on the ventral aspect of
the retina (data not shown).
Mass-specific blood volumes of –Hb icefishes are in the range of
7–9% of body mass, or approximately two- to fourfold greater than those
characteristic of most red-blooded fishes
(Kock, 2005). Some, but not
all, of this additional volume might be accounted for by the significantly
larger bore of blood vessels in icefishes than their red-blooded counterparts.
But, even this possibility is not valid for all tissues. For example, mean
capillary cross-sectional area in skeletal muscle of C. aceratus is
1.6-fold greater than that measured in N. coriiceps
(O'Brien et al., 2003).
Assuming a cylindrical geometry, this would correspond only to an identical
1.6-fold elevation in vascular volume of icefish skeletal muscle compared to
the tissue in red-blooded species, if vascular densities were equivalent in
both species; but they are not. Both capillary density and capillary length
density in pectoral muscle are lower in C. aceratus than in N.
coriiceps (O'Brien et al.,
2003) and it has been argued that instantaneous blood volume in
the microcirculation of icefish locomotory muscles is similar to that seen in
other teleosts (Egginton and Rankin,
1998). So, how can we account for accommodation of a greater
organismal volume of blood in the circulation of icefishes than red-blooded
species? At least part of the answer appears to be the proliferation of
vasculature seen in other tissues of icefishes.
Average length densities of retinal vessels from –Hb channichthyids
vary little from mean densities in +Hb bathydraconids that possess
intermediate Hcts, but are 1.5-fold greater than mean length densities
observed in +Hb nototheniids characterized by the greatest content of red
blood cells. VDI of retinal hyaloid arteries and the corresponding distance
separating them in the vascular array show a much more regular progression
(Table 2). Icefishes display
the greatest vascular densities and smallest intervessel distances, followed
by bathydraconid species, while nototheniids with the highest Hcts clearly
possess the lowest vascular densities and greatest intervessel distances.
Hb-deficient notothenioids thus maintain normal biological function in the eye
by compensations that include high densities of large-bore blood vessels and
short path lengths for oxygen diffusion.
The combination of larger bore and more densely distributed blood vessels
in –Hb icefishes also results in considerable increases in vascular
blood volume of the retina. For example, blood vessels of icefishes are at
least 1.5-fold larger in diameter than those of red-blooded notothenioids and
vascular densities of icefishes are up to 2.2 times greater. Thus, we can
anticipate that the instantaneous blood volume of retinal vasculature in
icefish species should be approximately fivefold greater than that of their
high-Hct +Hb relatives. Any other tissues that show similar differences in
vascular proliferation between –Hb and +Hb species likewise will
contribute significantly to disparities in organismal blood volume that have
been documented between icefishes and closely related red-blooded species.
Red blood cell content of +Hb species is directly related to vascular density
The general pattern of compensation described above is a logical one.
Aerobic demand of retinal tissue among the species studied is probably
similar, given that all are predominantly visual predators, based upon prey
items (Gon and Heemstra,
1990). Two possible compensatory mechanisms (or a combination of
both) may ensure uniform delivery of oxygen to retinas of animals with
decreasing blood oxygen-carrying capacities. First, elevation in the rate of
blood flow through vessels will maximize the driving PO2
gradient across the entire length of the exchange surface. This strategy is
most evident in the –Hb icefishes, as indicated by their much greater
cardiac outputs than observed in +Hb species. Although available data are
limited, the range of mass-specific cardiac outputs among the +Hb species is
relatively restricted (Axelsson,
2005). Absolute viscosity of blood containing red blood cells is
also greater than that of hemoglobinless species
(Wells et al., 1990).
Consequently, increasing the rate of flow of blood containing erythrocytes
through individual vessels would require significant elevation of vascular
pressures and does not appear to be a strategy employed by +Hb species. The
second possible mechanism for compensation of oxygen delivery is by anatomical
proliferation of the vascular array supplying the tissue, which both increases
the overall perfusion of the tissue and reduces the diffusional path length
for oxygen. We reasoned that vascular proliferation, therefore, must be the
predominant means of compensating for reductions in blood oxygen-carrying
capacity among the +Hb species. This prompted us to examine more closely the
relationship between red blood cell content and vascular densities among
red-blooded notothenioids in this study.
Although we were prepared to find a significant correlation between apparent capacity of blood to carry oxygen and anatomical indices for oxygen delivery to retinal tissue, we were surprised at how tightly linked theses features are among the red-blooded species. Across a >2.3-fold range in Hct, a decrease in erythrocyte content of the blood is met with both a compensatory proportional increase in density of blood vessels supplying the retina and a decrease in spacing between the blood vessels (Fig. 5A,B). In other words, the lower apparent oxygen content of blood, the greater the blood supply and shorter the diffusional distance for oxygen – a result that is somewhat satisfying from an intuitive sense. We might initially expect that extrapolation of these relationships to zero Hct would be predictive of values in the –Hb icefish species; yet, this is not the case. Icefish species consistently show lower vascular densities and wider intervessel distances than would be predicted. Upon further reflection, this is not really surprising, given very marked differences in parameters of blood flow between these groups. As mentioned earlier, one of the hallmark characteristics of channichthyid icefishes is their much greater cardiac output than observed in red-blooded relatives. A greater rate of vascular perfusion evidently diminishes the need for these compensations to provide adequate rates of oxygenation of retinal tissue.
What mechanism drives changes in vascular anatomy?
Our study has now added differences in vascular densities of a highly
oxidative tissue to a long list of cardiovascular disparities between
–Hb icefishes and their red-blooded relatives that appear to have been
driven by differences in oxygen-carrying capacity of their bloods. This
cause-and-effect relationship is further supported by the tight linkage
between vascular anatomy and erythrocyte content of the blood that we observed
among red-blooded notothenioids. Yet, the observations alone tell us nothing
about underlying mechanisms that yield the different anatomies. At least two
possible scenarios may be envisioned. First, these vascular characters may
have come about by the process of natural selection operating upon vascular
differences that arose through random mutations within the ancestral
populations of modern species. Such an explanation, however, begs the question
of how the dramatically altered vascular characters of icefishes could have
come about so rapidly in light of the relatively short evolutionary history of
this group of fishes [ca. 5 MYA (Near et
al., 2003)]. The alternative is that some type of physiological
feedback mechanism exists such that conditions arising from changes in the
level of erythrocytes containing oxygen-binding proteins affect vascular
development.
One of the most potent mechanical affectors of angiogenesis in vertebrate
animals is shear stress (Brown and Hudlicka, 2003). This rheological force is
proportional to both blood viscosity and velocity of flow and inversely
proportional to the radius of the blood vessel. Because hematocrit directly
affects viscosity, one might speculate that variance in hematocrits observed
among the red-blooded species in this study might lead to differences in shear
stress that could correlate with retinal vascular densities. It is impossible
to evaluate this conjecture rigorously because measurements of velocity of
blood flow through the retinas are unavailable. Our observations, however,
appear to be at direct odds with shear stress as a causative factor.
Reductions in hematocrit will result in decreased viscosity of blood, thus
lowering shear stress and presumably reducing this mechanical angiogenic
stimulus. Viscosity of whole blood in the hemoglobinless icefishes (Hct=0) is
substantially lower than that of their red-blooded counterparts
(Wells et al., 1990;
Egginton, 1996). Yet, we
observed increases in vascular proliferation with decreases in hematocrit
among the red-blooded fishes studied and the highest vascular density was
measured in a hemoglobinless icefish. On this basis, it seems reasonable to
discount shear stress as a major determinant of vascular densities in retinas
of Antarctic fishes. What then, might be the causative agent for vascular
proliferation?
We have recently suggested an alternative mechanism involving the
ubiquitous bioactive molecule, nitric oxide (NO)
(Sidell and O'Brien, 2006).
The most widely recognized role of NO is that of a potent vasodilator, but it
also is known to mediate a pathway that promotes the growth of capillary
networks (angiogenesis) (Conway et al.,
2001). NO is produced in vertebrate animal tissues in a reaction
catalyzed by a family of three isoforms of the enzyme nitric oxide synthase
(NOS). There is wide acceptance that the most quantitatively important
catabolic reaction of NO in vertebrate animals is its reaction with
oxyferrohemoglobin (oxyHb). Reaction of NO with oxyHb is very rapid and yields
products of metHb (methemoglobin) and nitrate; the reaction is considered to
be the major route of elimination of NO from the body and the major source of
circulating nitrate in vertebrates
(Kerwin, Jr et al., 1995).
Thus, reductions in levels of circulating Hb should lead to reduction in the
rate at which NO is degraded and elevation of its steady-state levels.
Elevated NO, in turn, could trigger vascular proliferation. The data we report
here are consistent with such an hypothesis.
If the mechanism described above proves to be correct, the implications are provocative. Such a mechanism suggests that notothenioids (like other vertebrates) intrinsically possess physiological feedback systems that may rapidly trigger changes in vascular density in response to altered concentration of circulating Hb. The logical extension of this is that physiology of ancestral channichthyids may have accelerated the evolution of compensations to the mutational loss of Hb expression and the development of their extreme cardiovascular characteristics.
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Abrams, D. C., Facer, P., Bishop, A. E. and Polak, J. M. (1994). A computer-assisted stereological quantification program: OpenStereo. Microsc. Res. Tech. 29,240 -247.[CrossRef][Medline]
Anctil, M. (1968). Intraocular vascular supply in some marine teleosts. Rev. Can. Biol. 27,347 -355.[Medline]
Axelsson, M. (2005). The circulatory system and its control. In Fish Physiology, Vol. 22, The Biology of Polar Fishes (ed. A. P. Farrel and J. Steffensen), pp.239 -280. San Diego: Elsevier Academic Press.
Brown, M. D. and Hudlika, O. (2003). Modulation of physiological angiogenesis in skeletal muscle by mechanical forces: involvement of VEGF and metalloproteinases. Angiogenesis 6,1 -14.[CrossRef][Medline]
Chase, J. (1982). The evolution of retinal vascularization in mammals. A comparison of vascular and avascular retinae. Ophthalmology 89,1518 -1525.
Conway, E. M., Collen, D. and Carmeliet, P.
(2001). Molecular mechanisms of blood vessel growth.
Cardiovasc. Res. 49,507
-521.
Copeland, D. E. (1976). The anatomy and fine structure of the eye in teleost. IV. The choriocapillaris and the dual vascularization of the area centralis in Fundulus grandis. Exp. Eye Res. 22,169 -179.[CrossRef][Medline]
Copeland, D. E. (1980). Functional vascularization of the teleost eye. Curr. Top. Eye Res. 3,219 -280.[Medline]
Eastman, J. T. (1988). Ocular morphology in Antarctic notothenioid fishes. J. Morphol. 196,283 -306.[CrossRef]
Eastman, J. T. (1993). Antarctic Fish Biology: Evolution in a Unique Environment. San Diego: Academic Press.
Eastman, J. T. (2005). The nature of the diversity of Antarctic fishes. Polar Biol. 28, 93-107.[CrossRef]
Eastman, J. T. and Lannoo, M. J. (2003). Diversification of brain and sense organ morphology in Antarctic dragonfishes (Perciformes: Notothenioidei: Bathydraconidae). J. Morphol. 258,130 -150.[CrossRef][Medline]
Eastman, J. T. and Lannoo, M. J. (2004). Brain and sense organ anatomy and histology in hemoglobinless Antarctic icefishes (Perciformes: Notothenioidei: Channichthyidae). J. Morphol. 260,117 -140.[CrossRef][Medline]
Eastman, J. T. and Lannoo, M. J. (in press). Brain and sense organ anatomy and histology of two species of phyletically basal non-Antarctic thornfishes of the Antarctic suborder Notothenioidei (Perciformes: Bovichtidae). J. Morphol. 268.
Egginton, S. (1996). Blood rheology of Antarctic fishes: viscosity adaptations at very low temperatures. J. Fish Biol. 48,513 -521.[CrossRef]
Egginton, S. and Rankin, C. (1998). Vascular adaptations for a low pressure/high flow blood supply to locomotory muscles of Antarctic icefish. In Fishes of Antarctica: A Biological Overview (ed. G. di Prisco, E. Pisano and A. Clarke), pp.185 -195. Milan: Springer-Verlag.
Fitch, N. A., Johnston, I. A. and Wood, R. E. (1984). Skeletal muscle capillary supply in a fish that lacks respiratory pigments. Respir. Physiol. 57,201 -211.[CrossRef][Medline]
Gon, O. and Heemstra, P. C. (ed.) (1990). Fishes of the Southern Ocean. Grahamstown: J. L. B. Smith Institute of Ichthyology.
Hanyu, I. (1962). Intraocular vascularization in some fishes. Can. J. Zool. 40, 87-106.
Hemmingsen, E. A. (1991). Respiratory and cardiovascular adaptations in hemoglobin-free fish: resolved and unresolved problems. In Biology of Antarctic Fish (ed. G. di Prisco, B. Maresca and B. Tota), pp. 191-203. New York: Springer-Verlag.
Hemmingsen, E. A. and Douglas, E. L. (1970). Respiratory characteristics of the hemoglobin-free fish Chaenocephalus aceratus. Comp. Biochem. Physiol. 33,733 -744.[Medline]
Hemmingsen, E. A., Douglas, E. L., Johansen, K. and Millard, R. W. (1972). Aortic blood flow and cardiac output in the hemoglobin-free fish Chaenocephalus aceratus. Comp. Biochem. Physiol. 43A,1045 -1051.[Medline]
Holeton, G. F. (1970). Oxygen uptake and circulation by a hemoglobinless Antarctic fish (Chaenocephalus aceratus Lonnberg) compared with three red-blooded Antarctic fish. Comp. Biochem. Physiol. 34,457 -471.[Medline]
Kawall, H. G., Torres, J. J., Sidell, B. D. and Somero, G. N. (2002). Metabolic cold adaptation in Antarctic fishes: evidence from enzymatic activities of brain. Mar. Biol. 140,279 -286.[CrossRef]
Kerwin, J. F., Jr, Lancaster, J. R., Jr and Feldman, P. L. (1995). Nitric oxide: a new paradigm for second messengers. J. Med. Chem. 38,4343 -4362.[CrossRef][Medline]
Knox, G. A. (1970). Antarctic marine ecosystems. In Antarctic Ecology. Vol.1 (ed. M. W. Holdgate), pp.69 -96. London: Academic Press.
Kock, K.-H. (2005). Antarctic icefishes (Channichthyidae): a unique family of fishes. A review, Part I. Polar Biol. 28,862 -895.[CrossRef]
Laws, R. M. (1985). The ecology of the Southern Ocean. Am. Sci. 73,26 -40.
Lewis, R. W. and Perkin, R. G. (1985). The winter oceanography of McMurdo Sound, Antarctica. In Antarctic Research Series, Vol. 43, Oceanology of the Antarctic Continental Shelf (ed. S. S. Jacobs), pp. 145-165. Washington: American Geophysical Union.
Littlepage, J. L. (1965). Oceanographic investigations in McMurdo Sound, Antarctica. In Antarctic Research Series, Vol. 5, Biology of the Antarctic Seas II (ed. G. A. Llano), pp. 1-37. Washington: American Geophysical Union.
Maas, J. W. M., Le Noble, F. A. C., Dunselman, G. A. J., de Goeij, A. F. P. M., Struyker Boudier, H. A. J. and Evers, J. L. H. (1999). The chick embryo chorioallantoic membrane as a model to investigate the angiogenic properties of human endometrium. Gynecol. Obstet. Invest. 48,108 -112.[CrossRef][Medline]
Near, T. J., Pesavento, J. J. and Cheng, C.-H. (2003). Mitochondrial DNA, morphology and the phylogenetic relationships of Antarctic icefishes (Notothenioidei: Channichthyidae). Mol. Phylogenet. Evol. 28, 87-98.[CrossRef][Medline]
Nicol, J. A. C. (1989). The Eyes of Fishes. Oxford: Oxford University Press.
O'Brien, K. M., Skilbeck, C., Sidell, B. D. and Egginton, S.
(2003). Muscle fine structure may maintain the function of
oxidative fibres in haemoglobinless Antarctic fishes. J. Exp.
Biol. 206,411
-421.
Rieder, M. J., O'Drobinak, D. M. and Greene, A. S. (1995). A computerized method for determination of microvascular density. Microvasc. Res. 49,180 -189.[CrossRef][Medline]
Ruud, J. T. (1954). Vertebrates without erythrocytes and blood pigment. Nature 173,848 -850.[CrossRef][Medline]
Sidell, B. D. and O'Brien, K. M. (2006). When
bad things happen to good fish: the loss of hemoglobin and myoglobin
expression in Antarctic icefishes. J. Exp. Biol.
209,1791
-1802.
Strick, D. M., Waycaster, R. L., Montani, J. P., Gay, W. J. and Adair, T. H. (1991). Morphometric measurements of chorioallantoic membrane vascularity: effects of hypoxia and hyperoxia. Am. J. Physiol. 260,H1385 -H1389.[Medline]
Voss, K., Jacob, W. and Roth, K. (1984). A new image analysis method for the quantification of neovascularization. Exp. Pathol. 26,155 -161.[Medline]
Walls, G. L. (1942). The Vertebrate Eye and its Adaptive Radiation, Bulletin No. 19. Bloomfield Hills, MI: Cranbrook Institute of Science.
Waser, W. P. and Heisler, N. (2004). Oxygen delivery to the fish eye: blood flow in the pseudobranchial artery of rainbow trout (Oncorhynchus mykiss). Fish Physiol. Biochem. 30,77 -85.[CrossRef]
Weibel, E. R. (1979). Stereological Methods: Practical Methods for Biological Morphometry. Vol.1 . New York: Academic Press.
Wells, R. M. G., Ashby, M. D., Duncan, S. J. and Macdonald, J. A. (1980). Comparative study of the erythrocytes and haemoglobins in nototheniid fishes from Antarctica. J. Fish Biol. 17,517 -527.[CrossRef]
Wells, R. M. G., Macdonald, J. A. and di Prisco, G. (1990). Thin-blooded Antarctic fishes: a rheological comparison of the haemoglobin-free icefishes Chionodraco kathleenae and Cryodraco antarcticus with a red-blooded nototheniid, Pagothenia bernacchii. J. Fish Biol. 36,595 -609.[CrossRef]
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