Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

Podsumowanie

We present a non-invasive ultrasound technique for generating three-dimensional angiographies in the eye without the use of contrast agents.

Streszczenie

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood supply. The capillary lamina of the choroid lines the outer surface of the retina and is the dominating source of oxygen in most vertebrate retinas. However, this vascular bed is challenging to image with traditional optical techniques due to its position behind the highly light-absorbing retina. Here we describe a high-frequency ultrasound technique with subsequent flow-enhancement to image deep vascular beds (0.5-3 cm) of the eye with a high spatiotemporal resolution. This non-invasive method works well in species with nucleated red blood cells (non-mammalian and fetal animal models). It allows for the generation of non-invasive three-dimensional angiographies without the use of contrast agents, and it is independent of blood flow angles with a higher sensitivity than Doppler-based ultrasound imaging techniques.

Wprowadzenie

The high metabolism on the vertebrate retina imposes an intrinsic tradeoff between two contrasting needs; high blood flow rates and a light path devoid of blood vessels. To avoid visual disturbance of perfusing red blood cells, the retina of all vertebrates receives oxygen and nutrients via a sheet of capillaries behind the photoreceptors, the choriocapillaris^1,2,3. However, this single source of nutrients and oxygen imposes a diffusion limitation to the thickness of the retina^4,5, so many visually active species possess a variety of elaborate vascular networks to provide additional blood supply to this metabolically active organ⁶. These vascular beds include blood vessels perfusing the internal retinal layers in mammals and some fishes^4,7,8,9,10, blood vessels on the inner (light-facing) side of the retina found in many fishes, reptiles, and birds^4,11,12,13, and countercurrent vascular arrangements of the fish choroid, the choroid rete mirabile, that allows for the generation of super-atmospheric oxygen partial pressures^{14,15,16,17,18,19,20}. Despite that these additional non-choroidal paths for retinal nutrient supply play an essential role in fueling the metabolic requirements of superior vision⁴, the three-dimensional anatomy of these vascular structures is poorly understood, limiting our understanding of the morphological evolution of the vertebrate eye.

Traditionally, retinal blood supply has been studied using optical techniques, such as fundus ophthalmoscopy. This category of techniques provides high-throughput non-destructive information on non-choroidal blood vessel anatomy in high-resolution²¹ and is therefore readily used in clinical diagnosis of abnormalities in retinal vessel structure²². However, the retinal pigment epithelium absorbs the transmitted light and limits the depth of view in these optical techniques, providing reduced information on choroidal structure and function without the use of contrast agent²¹. Similar depth limitations are experienced in optical coherence tomography (OCT). This technique can generate high-resolution fundus angiographies using light waves at the technical expense of depth penetration²³, while the enhanced depth imaging OCT can visualize the choroid at the expense of retinal imaging quality²⁴. Magnetic resonance imaging overcomes the optical limitations of ophthalmoscopy and OCT and can map vascular layers in the retina, albeit at a low resolution²⁵. Histology and microcomputed tomography (µCT) maintain the high-resolution of the optical techniques and provide information on whole-eye vascular morphology⁴, but both techniques require ocular sampling and are therefore not possible in the clinic or rare or endangered species. To overcome some of the limitations of these established retinal imaging techniques, the study here presents an ultrasound protocol on anesthetized animals, where blood movement is mapped in silico on a series of equally-spaced two-dimensional ultrasound scans spanning a whole eye by applying a comparable technique as described previously for embryonic and cardiovascular imaging^26,27,28 and in OCT angiography²⁹. This approach allows for the generation of non-invasive three-dimensional deep ocular angiographies without using a contrast agent and opens up new avenues for mapping blood flow distribution within the eye across species.

Protokół

The protocol below was performed with permission from the Danish Inspectorate for Animal Experimentation within the Danish Ministry of Food, Agriculture, and Fisheries, Danish Veterinary and Food Administration (Permit number 2016-15-0201-00835).

1. Anesthesia and ultrasound medium

1. Anesthetize the research animal.
   NOTE: Type and dose of appropriate anesthesia are highly species-dependent. In general, immersion-based anesthetics such as MS-222 (ethyl 3-aminobenzoate methanesulfonic acid), benzocaine (ethyl 4-aminobenzoate), and propofol (2,6-diisopropyl phenol) are useful in fish and amphibians which readily absorbs the anesthetic over gills or skin (e.g., 0.05 mg·L^-1 benzocaine in rainbow trout). A range of dissolved compounds that can be administered intravenously, intramuscularly, intraperitoneally is available for amniotes, as are gas-based anesthetics. Alfaxalon administered intramuscularly is useful in reptiles (e.g., 30 mg·kg^-1 in lizards), and isoflurane administered as gas is useful in birds (e.g., 2% in air for pigeons). Refer to the published literature^30,31,32 for a full overview of available anesthetics across species.
2. Test reflexes in the animal to confirm an optimal level of anesthesia. Ensure that the animal is completely motionless during the procedure as the flow-enhanced ultrasound procedure is sensitive to motion noise.
   1. Too deep anesthesia can alter blood flow patterns, so conduct a dose titration in the start-up phase of an experiment.
   2. Increase the anesthesia dosage in steps and observe blood flow in the eye aided by simple brightness mode (B-mode) ultrasound.
      NOTE: An optimal level of anesthesia is obtained when the animal is motionless (except respiration) with visible ocular blood flow.
3. If the type/dose of anesthetic is not permissive for respiratory movements, then ensure adequate ventilation of the animal, e.g., using an air pump to oxygenate the water for aquatic species or a ventilator for air-breathing species.
4. Position the animal in a posture that allows direct access from above to the eye.
   NOTE: Depending on species, this can be in either a supine or lateral position. It can be useful to construct a simple holding device using a small piece of non-reactive metal (e.g., stainless steel) and loose rubber bands (see Figure 1).
5. Place appropriate ultrasound medium on the eye of the animal. If scaled eyelids (ultrasound impermeable) cover the eye, then displace these gently with a cotton swab.
   ​NOTE: For aquatic species, the best ultrasound medium is clean tank water in which the animal usually lives. For terrestrial species, a generous amount of ultrasound gel ensures free movements and imaging of the ultrasound transducer (i.e., linear array probe) across the entire surface of the eye. Vet ointment on the contralateral eye is required for terrestrial species.

2. 2D and 3D ocular ultrasound image acquisition

1. Position the ultrasound transducer medial to the eye in either a dorsal/ventral or rostral/caudal orientation depending on desired image orientation.
2. In B-mode, with a maximum depth of field, image the medial and deepest portion of the eye and make sure that all structures of interest are visible in the image field.
   NOTE: In some species, the crystalline lens takes up a comparatively large proportion of the vitreous humor, which may absorb the ultrasound, especially at higher frequencies.
3. Slowly translate the transducer to each side while inspecting the real-time images. Make sure all structures of interest are visible in the image field; if not, switch to a transducer with a lower frequency and larger depth of field.
   NOTE: The following center frequencies allow for the following maximum depth of field: 21 MHz: 3 cm, 40 MHz: 1.5 cm, 50 MHz: 1 cm (see Table 1). However, these maximum depth of field values can be markedly lower if the eye contains calcified or other ultrasound impermeable structures.
4. Adjust image depth, depth offset (distance from the top of the image to the structure of interest), image width, as well as number and position of focal zones to cover the desired region of interest in all three spatial dimensions (e.g., 1 cm image depth, 2 mm depth offset, 1 cm image width, one focal zone).
   NOTE: Although specific naming of buttons that adjust these parameters may vary between ultrasound systems, most systems will have buttons with logical names for these adjustments. These image parameter settings usually affect the range of possible temporal resolutions of the ultrasound acquisition.
5. Set frame rate in the range of 50-120 frames·s^-1.
   NOTE: The temporal resolution (i.e., the time interval between successive B-scans) must be adequate to display large pixel intensity variability in imaged blood vessels, i.e., the temporal resolution must not be too high. On the other hand, to complete a full 3D recording of the eye in a reasonable time, temporal resolution cannot be too low. A temporal resolution ranging from 50-120 frames·s^-1 is usually adequate for the flow-enhanced procedure in most species. On some ultrasound systems, this desired temporal resolution can be obtained by switching between the "general imaging" (high spatial/low temporal resolution) and "cardiology" (low spatial/high temporal resolution) modes.
6. Adjust 2D gain to a level (~5 dB), so anatomical structures are only just visible in the B-mode acquisition to increase the signal-to-noise ratio in the subsequent flow-enhanced reconstruction.
7. To acquire a 2D flow-enhanced image at a single slice position, translate the transducer to this position and continue at step 3.1.
8. To acquire a 3D recording of an entire region of interest, e.g., the retina, translate the transducer to one extreme of the region of interest.
   1. To determine the exact position of the extreme end of the region of interest, increase the 2D gain briefly.
   2. After correct transducer placement is complete, lower the 2D gain before recording to ensure maximal signal-to-noise ratio in the subsequent flow-enhanced reconstruction.
9. For each step (slice) in the 3D recording, acquire ≥100 frames (optimally ≥1000 frames).
10. Using a micromanipulator or build-in transducer motor, translate the transducer across the entire region of interest in steps of, e.g., 25 µm or 50 µm (remember to note the step size) and repeat the ≥100 frames acquisition for each step.
11. Euthanize the research animal according to the animal care guidelines of the institution.

3. Flow-enhanced image reconstruction

1. Export the recordings into digital imaging and communications in medicine (DICOM) file format (little-endian).
2. To produce a single flow-enhanced image based on a ≥100 frames (T) cine recording, calculate the standard deviation on pixel level (STD(x,y)) using the formula:
   
   Where I_t(x,y) is the intensity of the pixel at the (x,y) pixel coordinate at time t, and Ī_t(x,y) is the arithmetic mean value of I over time.
3. Repeat step 3.2 for each slice in the 3D recording.
4. To automate the STD-calculation and image reconstruction process for multiple slices in a 3D recording, conduct this operation in batch mode using, e.g., ImageJ and the supplementary macro script (Supplementary File 1).
5. Combine all reconstructed slices into one image stack (Images to Stack command in ImageJ).
6. Specify slice thickness from the step size used during acquisition (Properties command in ImageJ).
7. Save the image stack as a 3D TIF file.
   NOTE: Flow-weighted three-dimensional recordings of ocular blood vessels can subsequently be used to create volume renderings and build digital and physical anatomical models of vascular structures of the eye. These image processing options are outside the scope of this protocol; refer to the previously published articles for more details^33,34,35.

Wyniki

The flow-enhanced ultrasound technique to image vascular beds of the eye can be applied in a range of species and has currently been used in 46 different vertebrate species (Figure 1, Table 1). The presence of nucleated red blood cells in non-adult-mammalian vertebrates provides positive contrast of flowing blood compared to static tissue in cine recordings (Supplementary File 2). However, when analyzed on a frame-by-frame basis, the clear distinction betwee...

Dyskusje

Vascular imaging using flow-enhanced ultrasound provides a new method for non-invasive imaging of the vasculature of the eye that offers several advantages over present techniques but has its intrinsic limitations. The primary advantage of flow-enhanced ultrasound is the ability to generate ocular angiographies with a depth of field that exceeds the retinal pigment epithelium, which limits the depth of field in optical techniques. In ultrasound imaging, spatial resolution and depth of field are ultimately determined by t...

Materiały

Name	Company	Catalog Number	Comments
MS-222	Sigma	E10521-50G
Benzocaine	Sigma	E-1501
Propofol	B Braun	12260470_0320
Alfaxalon	Jurox	NA
Isoflurane	Zoetis	50019100
Ultrasound scanner	VisualSonics	Vevo 2100