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Keywords:

  • CT;
  • vessel imaging;
  • barium sulfate;
  • advanced visualization;
  • 2DTF

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

The Brazilian free-tailed bat (Tadarida brasiliensis) exhibits a highly vascularized, hairless thermal window (or “radiator”) on the proximal ventral surfaces of extended wings and body. We identified this character using thermal infrared imaging and investigated the vasculature using barium sulfate enhanced microcomputed tomography (micro-CT). Micro-CT images revealed unique arrangements of arteries and veins in the region of the radiator positioned perpendicular to the axis of the body. Coupling micro-CT imaging with analysis of surface temperature profiles, we concluded that radiators aid in thermoregulation during flight in variable environments. This study represents the first application of contrast enhanced micro-CT to visualize vasculature of bats and thus exhibits a promising technique for further investigations of cardiovascular function and anatomy in bats. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Free-tailed bats (Order: Chiroptera; Family: Molossidae) are abundant in the warmer parts of the world and are specialized for high-altitude flight and hawking foraging behavior (Vaughan et al., 1966). One species, the Brazilian free-tailed bat (Tadarida brasiliensis), encounters highly variable thermal environments while transitioning between low altitudes as they emerge from and return to roosts and high altitudes during prolonged flight while commuting and foraging. In south-central United States, this species often emerges before sunset when ambient temperatures exceed 35°C, but shortly thereafter, experiences high radiative heat loss to the night sky (Reichard et al.,2010a). Several physiological and morphological adaptations facilitate regulating euthermic body temperature to match such behaviors (Schmidt-Nielsen et al.,1990). Thermal infrared (TIR) imaging has been used for censusing maternity colonies of this and other species (Sabol et al.,1995; Betke et al.,2008) and for studying roosting and foraging behaviors of Brazilian free-tailed bats (Hristov et al.,2008). TIR imaging has also been used to characterize surface temperatures of different body regions of Brazilian free-tailed bats, and revealed unique, radiator-like vascular morphology that serves as a thermal window beneath the proximal section of the wings and adjacent body (Reichard et al.,2010b).

Over the past decade, contrast enhanced, high-resolution computed tomography (micro-CT) imaging has been regularly used to investigate vessel development in disease models (Duvall et al.,2004) and to enhance the visualization of vasculature and organ systems in small animals (Ritman et al.,2005; Mulder et al.,2006; Prajapati et al.,2011). We used this method to investigate circulatory patterns in the radiators of Brazilian free-tailed bats and to identify the origins and destinations of arteries and veins that serve the radiators. To do so, we used barium sulfate (BaSO4) enhanced micro-CT imaging. In addition to describing a unique organ, this study represents the first application of contrast enhanced micro-CT in bats.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

All procedures used in this study were in accordance with the American Society of Mammalogists Guidelines for Capture, Handling, and Care of Mammals (Sikes et al., 2011), Boston University's Institutional Animal Care and Use Committee, and Texas Department of Parks and Wildlife.

TIR Imaging

TIR imaging studies were conducted between 2006 and 2009 during the summer at two caves in south-central Texas; Frio Cave, Uvalde County and Eckert James River Cave, Mason County. TIR images of Brazilian free-tailed bats were recorded with a Merlin Mid TIR camera configured with a 25-mm (22°) lens (Indigo Systems, Santa Barbara, CA). This camera was specifically calibrated by the manufacturer for temperatures and distances used in this study (precision 0.018°C, accuracy ± 2°C). TIR Images were captured in 1.0–5.4 μm wavelength range and TIR digital videos were analyzed using Rtools-Rview thermal image analysis software (FLIR Systems, Boston, MA).

Contrast Enhanced Micro-CT Imaging

We examined the vasculature of radiators on Brazilian free-tailed bats using a contrast enhanced micro-CT technique. Bats were lightly anesthetized with isoflorane and injected with 200 μL of BaSO4 (100% weight per weight (wt/wt) and 56% weight per volume (wt/vol); BaSO4) intravenously through the interfemoral vein. These bats were then euthanized without regaining consciousness and scanned at 93 μm isometric resolution using an eXplore Locus RS Small Animal Micro-CT Scanner (GE Healthcare, London, Ontario). Images were reconstructed using the manufacturer's proprietary EVSBeam™ software and analyzed using visualization tools MicroView™ and ImageVis3D.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

TIR Imaging Reveals Hot-Spots on Brazilian Free-Tailed Bats

TIR imaging revealed distinct hot spots beneath the proximal portion of the wings parallel to the flanks of free-ranging Brazilian free-tailed bats from the antebrachial to the pelvic region (Fig. 1A). Surface temperatures along a profile from wing membrane to the midline of the body showed distinct higher-temperature peaks corresponding to the location of radiators (Fig. 1A inset). Visual inspection of these hot spots exposed a region of highly-vascularized, hairless skin along the ventral margin of the wing and body that has been described in detail in a related study (Reichard et al.2010b). This region of the body was warmer than surrounding areas and exhibited common characteristics of a thermal window that is exposed when wings are fully abducted during flight.

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Figure 1. Visualization and characterization of the vasculature of a Brazilian free-tailed bat (Tadarida brasiliensis). (A) TIR image of a Brazilian free-tailed bat emerging from a cave. The white line on the bat represents the profile of surface temperature that corresponds with the plotted surface temperature (inset). The white arrow on the TIR image corresponds with the black arrow on the Ts plot. The thermal bar (on the right) ranges from 0 to 42°C. (B) Radio-opaque contrast enhanced micro-CT imaging of a Brazilian free-tailed bat. Maximum intensity projection (MIP) image showing blood vessels perfused with BaSO4. The bright slug-shaped structure in the bat's abdomen in the MIP image represents the partially digested exoskeletons of insects in the bat's gut. Inset shows zoomed in view of the radiator region. (C) Two-dimensional transfer function (2DTF) rendering created in Imagevis3D software using the BaSO4 enhanced micro-CT data. Red: BaSO4 (blood vessels), Brown: skin, Beige: Skeleton. BaSO4 was injected into the interfemoral (IF) vein, which is visible in both images (B, C) on the tail membrane between the bones of the leg and tail. Radiator vessels are indicated by yellow arrow heads in both images. UL- ulnar artery, SCI- superficial circumflex iliac vein, IF- interfemoral vein.

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Micro-CT Imaging Helps Visualize and Characterize Blood Vessels

Radio-opaque BaSO4 solution injected to the interfemoral vein in the tail membrane perfused arteries and veins of the body, head, forelimbs, hindlimb, and tail. The Maximum Intensity Projection (MIP; Fig. 1B) and two-dimensional transfer function (2DTF) images (Fig. 1C) showed the unique radiator as an arrangement of arteries and veins that are positioned perpendicular to the axis of the body and adjacent to the proximal region of the wing and flanks. Although distal arterioles and venules on the wing showed clear perfusion, not all of the radiator vessels were sufficiently perfused to appear in the images. We believe this outcome is due to altered circulation through various blood vessels while the bat was held (i.e., not flying) and anethesized before injection (Kallen et al., 1977).

Images processed with MIP and 2DTF rendering exhibited vascular anatomy in situ with skeletal features and other tissues (Fig. 1B,C). Radiator vessels (indicated by yellow arrow heads in Fig. 1B,C) are oriented perpendicular to the long axis of the body and are connected to larger vessels lying exactly on the ventral margin of the wing and body. Around the mid-axis of the wing, an arc is formed by paired arteries and veins of the ulnar and superficial circumflex iliac branches (Fig. 1C). To our knowledge, this unique vascularity associated with the radiator in Brazilian free-tailed bats has not been previously described.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

Brazilian free-tailed bats move rapidly from conditions requiring heat dissipation to avoid hyperthermia to conditions where conservation of heat may be essential. Radiators discovered with TIR imaging represent thermal windows similar to those known in several other animals. For example, four species of seals exhibit thermal windows as warm patches of skin, which are likely related to increased circulation through insulating blubber to the body surface (Mauck et al.,2003; Norris et al.,2010). Several waterfowl species also regulate blood flow through their bills to control body temperature during flights (Scott et al.,2008). Based on micro-CT scans of bats with BaSO4 injected into the bloodstream, coupled with analysis of thermal profiles, we concluded that uninsulated radiators in this species can be best described as a high density of blood vessels located in the proximal portion of the wing and along the flanks of the body. During powered flights, the radiator region is exposed when wings are outstretched and flapping, facilitating dry (sensible) heat loss to the surrounding environment. Moreover, regulated blood flow through radiators and the rest of the wings permits rapid dissipation of heat to avoid hyperthermia under environmental conditions where thermogenesis could otherwise exceed heat loss. Bar-headed geese utilize thermal windows to depress body temperature while flying in hypoxic conditions at high altitudes (Scott et al.,2008). Because Brazilian free-tailed bats are known to fly as high as 3000 m (Williams et al.,1973), radiators may similarly facilitate depression of Tb. The radiator appears to be a unique trait in the family Molossidae that may facilitate energy and water balance during sustained, high-altitude dispersal and foraging flights. Reichard et al. (2010b) investigated occurrence of radiator-like morphology in preserved specimens of more than 100 species representing 15 chiropteran families and determined that the combination of vasculature and hairless regions appeared only in molossids. Notwithstanding, further examination of living specimens is needed to corroborate these taxon-level comparisons.

Contrast enhanced micro-CT and infrared thermography of radiators in Brazilian free-tailed bats offer a promising approach to study the function of this structure under different physiological and environmental conditions. No previously published studies were identified in which contrast enhanced micro-CT imaging was used to study bats. This technique together with TIR imaging identified unique thermal windows in Brazilian free-tailed bats and offers a novel approach for investigating gross vascular anatomy of intact bats. Vascular contrast enhanced micro-CT imaging has been routinely used in clinical environments and holds great promise for diverse applications of research on small animals in both laboratory and field studies.

This approach could also be used in scanning live animals to characterize circulation patterns and fine vasculature (Prajapati et al.,2011). As a potential application, contrast-enhanced micro-CT could be used to explore tissue damage and healing ability in bats whose wings have been damaged by fungal infections associated with white-nose syndrome (WNS) (Reichard and Kunz,2009; Fuller et al.,2011). Current hypotheses about the progression of WNS suggest fungal infections may alter circulation-related physiological processes during hibernation (Cryan et al.,2010) and that healed wing tissue may have compromised physiological functions (Fuller et al.,2011).

Small animal micro-CT scanners are widely available at major imaging facilities. Based on the location and type of scanner, a scan with similar settings can cost around $80-$200 per animal. This technique can be used with any species with the contrast agent dose adjusted in proportion to the total blood volume of the animal being investigated. Because preclinical micro-CT units have gantry bore diameters of 8–10 cms, this potentially limits the size of species that can be scanned. However for larger animals, clinical CT scanner can be used with gantry bore sizes between 60 and 90 cm.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED

The authors thank the Nature Conservancy for access to Eckert James River Bat Cave and I. Marback and W. Cofer for access to Frio Cave. They also thank L. Allen, S. Austad, A. Bahadur, C. Casey, B. French, L. Gonzalez, N. Hristov, and L. Sloan for the assistance with field and lab work.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
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