This work has received funding from the Carlsberg Foundation (CF17-0778; CF18-0658), the Lundbeck Foundation (R324-2019-1470; R346-2020-1210), the Velux Foundations (00022458), The A.P. M&#248;ller Foundation for the Advancement of Medical Science, the European Union&#39;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement (No. 754513), and The Aarhus University Research Foundation.

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
<ol>
	<li>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 methanesu...

<ol>
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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...

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 choriocapillaris1,2,3. However, this single source of nutrients and oxygen imposes a diffusion limitation to the thickness of the retina4,5, 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 fishes4,7,8,9,10, blood vessels on the inner (light-facing) side of the retina found in many fishes, reptiles, and birds4,11,12,13, and countercurrent vascular arrangements of the fish choroid, the choroid rete mirabile, that allows for the generation of super-atmospheric oxygen partial pressures14,15,16,17,18,19,20. 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 (&#181;CT) 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 imaging26,27,28 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MS-222</td>
			<td>Sigma</td>
			<td>E10521-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine</td>
			<td>Sigma</td>
			<td>E-1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>B Braun</td>
			<td> 
			12260470_0320</td>
			<td></td>
		</tr>
		<tr>
			<td>Alfaxalon</td>
			<td>Jurox</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>50019100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound scanner</td>
			<td>VisualSonics</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
	</tbody>
</table>

deep vascular imaging in the eye with flow-enhanced ultrasound

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.

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...

Watch this Scientific Journal Video about Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound at JoVE.com

Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

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

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 ...

The Flow-Enhanced Ultrasound technique allows us to image the vasculature of the eye in three dimensions, without use of contrast agents. The main advantage of this technique is its stability to image the vesture, behind the pigmented retina, which is challenging with several optical imaging techniques. We demonstrate this technique in goldfish but it can be applied to all species with nucleated red blood cells, allowing researchers to gain insight into the eyes functional evolution.This technique can be applied by researchers with basic training in ultrasound imaging, and animal handling. After confirming an optimal level of anesthesia, position the animal in a posture that allows direct access from above, to the eye. Place a suitable ultrasound medium on the eye of the animal.If scaled eyelids cover the eye, then, displace these gently with a cotton swab. For aquatic animals, water works well as an ultrasound medium, Next, position the ultrasound transducer, medial to the eye, in either a dorsal ventral or rostral caudal orientation, depending on desired image orientation. In B-Mode, with a maximum depth of field, image the medial and deepest portion of the eye, to make sure that all structures of interest are visible in the image field.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.Adjust image depth, depth offset, image width, and number of position of focal zones, to cover the desired region of interest in all three spatial dimensions. Then, set frame rate in the range of 50 to 120 frames per second. Next, adjust the 2D gain to a level, such that the anatomical structures are only just visible in the B-Mode acquisition to increase the signal to noise ratio in the subsequent flow-enhanced reconstruction.To acquire a 2D flow-enhanced image at a single slice position, translate the transducer to this position, and proceed to flow-enhanced image reconstruction. To acquire a 3D recording of an entire region of interest, translate the transducer to one extreme of the region of interest. To determine the exact position of the extreme end, increase the 2D gain, briefly.After the transducer is placed correctly, lower the 2D gain before recording, to ensure maximal signal to noise ratio in the subsequent flow-enhanced reconstruction. For each step or slice in the 3D recording, acquire greater than 100 frames. Then, using a micro manipulator or built-in transducer motor, translate the transducer across the entire region of interest, in steps of either 25 or 50 micrometers, and repeat the acquisition of more than 100 frames for each step.For flow-enhanced imagery construction, export the recordings into the DICOM file format. To produce a single flow-enhanced image based on a greater than 100 frames scene recording, calculate the standard deviation on a pixel level using this formula, and repeat the calculation for each slice in the 3D recording. To automate the standard deviation calculation and image reconstruction process for multiple slices in a 3D recording, conduct this operation in batch mode using ImageJ and the macro script, provided in the text manuscript.Combine all reconstructed images into one image stack using the Images to Stack command in ImageJ. Then, using the Properties command, specifies slice thickness from the step size used during acquisition, and save the image stack as a 3D TIFF file. The presence of nucleated red blood cells in non-ad adult mammalian vertebrates, provides positive contrast of flowing blood, compared to static tissue in scene recordings.However, when now analyzed frame by frame, the clear distinction between blood and surrounding tissue is less obvious. This blood flow-enhancement procedure, essentially, compiles a multi-time point recording in 2D space, into a single image, where in the inherent signal value fluctuation in pixels, positioned in flowing blood, score a higher standard deviation, than surrounding static tissue, producing positive contrast. In 3D acquisitions, multiple parallel slices with known spacing can be combined into 3D image data.It can be used for three dimensional volume rendering and anatomical modeling. Doppler based ultrasound imaging also provides the option to specifically image blood flow, however, with less sensitivity than this method. This flow-enhanced ultrasound procedure allows for blood flow imaging in a range of species with nucleated red blood cells.Deep ocular vascular beds, such as the choroid rete mirabile in some fish, can be imaged if present in the species. The method is limited by the absence of nucleated red blood cells in mammals, in which the flow-enhancement procedure produces some degree of blood flow contrast, but not as distinct as in species with nucleated red blood cells. Flow-enhanced ultrasound is sensitive to motion noise.Respiratory movements can cause image blurring and artifacts, such as tissue border enhancement. Prospective or retrospective gating can be used to adjust for motion noise. For both 2D and 3D acquisitions, it is crucial to limit motion artifacts caused by the animal moving, due to inadequate anesthesia on unstable transducer set up.The development of this technique, paved the way for non-invasive in vivo examination of deep vascular networks in the eye of the vast majority of vertebrates, which possess nucleated erythrocytes.

deep-vascular-imaging-in-the-eye-with-flow-enhanced-ultrasound

Research

JoVE Journal

Biology

2.2K visualizações.  Aarhus University. A técnica de Ultrassom Aprimorado por Fluxo nos permite imaginar a vasculatura do olho em três dimensões, sem o uso de agentes de contraste. A principal vantagem dessa técnica é sua estabilidade para a imagem da vesture, por trás da retina pigmentada, que é desafiadora com várias técnicas de imagem óptica. Demonstramos essa técnica em peixes dourados, mas ela pode ser aplicada a todas as espécies com glóbulos vermelhos nucleados, permitindo que os pesquisadores obtenham uma visã...