Intravital optoacoustic ultrasound bio-microscopy reveals radiation-inhibited skull angiogenesis

Hector Estrada, Johannes Rebling, Wolfgang Sievert, Daniela Hladik, Urs Hofmann, Sven Gottschalk, Soile Tapio, Gabriele Multhoff, Daniel Razansky

Posted on: 13 May 2019 , updated on: 14 May 2019

Preprint posted on 19 February 2019

Article now published in Bone at http://dx.doi.org/10.1016/j.bone.2020.115251

Shedding light on skull and cerebral vascular changes: optoacoustic ultrasound bio-imaging shows irradiation effects on angiogenesis inhibition.

Selected by Mariana De Niz

Categories: biophysics, cancer biology, pathology, physiology

Background

Optical microscopy has been pivotal for biological discovery for more than three centuries. However, microscopy methods are limited for in vivo tissue imaging by light scattering. Scattering consists on photon absorption and re-emission without loss of energy, but with a change in photon direction which occurs due to photon interaction with cellular structures. Multiple scattering events result in photon diffusion. This ultimately imposes limits to the penetration capacity of microscopic imaging. Additionally, different tissues have different absorption and scattering coefficients, making some tissues more or less amenable to optical imaging techniques (Reviewed in 1).

Various imaging methods have been established to overcome these limitations. Among them is optoacoustic ultrasound imaging, which has been pioneered and implemented for various biological questions by a handful of groups worldwide, including Prof. Daniel Razansky’s (Figure 1A, B). In the work presented here, Estrada et al (2) use optoacoustic ultrasound bio-microscopy to study radiation-induced damage in the skull bone marrow and microvasculature, following radiotherapy (Figure 1C).

The calvarian bone marrow is an important site for blood and immune cell generation and is maintained by the complex microvasculature network, composed by the calvarian sinusoids. Damage to this vascular network is clinically relevant for homeostasis, as the sinusoids provide an interface between the hematopoietically active bone marrow and the peripheral circulation.

The study sheds light on vascular changes upon radiotherapy, previously poorly understood, but which has important translational relevance, as radiotherapy is commonly used for cancer treatment.

Figure 1: (A) Setup. Imaging of skull and brain vasculature (B) was performed by focusing nanosecond laser pulses with a custom-designed gradient index (GRIN) lens and detecting the generated optoacoustic responses by the same transducer used for the US reflection-mode imaging. (C) Irradiation of half of the skull resulted in inhibited angiogenesis in the calvarium microvasculature (blue) of the irradiated hemisphere, but not the non-irradiated one. Adapted from (2).

Key findings

In this work, Estrada et al used a hybrid imaging approach based on optoacoustic and ultrasound bio-microscopy. This approach allowed imaging over 6mm across the skull, with a spatial resolution of 12um and 30um in lateral and axial dimensions.
The work introduces a novel segmentation method that allows differentiation of the calvarian vasculature based on its elastic and structural properties. This enabled distinguishing this microvascular network from the cerebral one.
At 11 weeks following radiotherapy on half of the skull, key differences were noted between radiated and irradiated hemispheres: the sinusoidal vascular network in the calvarium remained intact only in the non-radiated hemisphere, while vasculature in the irradiated hemisphere (20 Gy) did not develop (Figure 1C).
Quantitative analysis of vascular changes showed not only a decrease in number of vessels in irradiated hemispheres, but also a decrease in the number of branch points detected in the vascular network, suggesting altogether a loss of complexity in the vascular network.

Open questions and what I like about this paper

I liked this paper because of the usefulness of the tools developed by the authors, and the translationally relevant aspect of the findings, to vascular, and cancer research.

Given the non-invasive nature of the method, and since you mention previous links to radiation-induced long-term cognitive disability, would it be possible in the future, to combine this method with other imaging platforms such as functional MRI, to study the direct link of angiogenesis inhibition, with collateral effects of irradiation?
In the past, your lab has generated hybrid systems combining fluorescent and optoacoustic methods. Could you combine your findings at mesoscopic level with fluorescence methods to image hematopoietic niche changes, and immune cell dynamics derived from damage to the calvarian microvasclature after irradiation?
Your study focused on the effects of irradiation on vascular remodelling, and you discuss effects in the context of cancer treatment. Your hybrid imaging method to visualize large areas of the skull, and to differentiate between cerebral and calvarian bone marrow could be applied to study other pathologies where changes to both compartments are induced. Have you considered applying this to other research topics?
Given the possibility to differentiate calvarian from cerebral vasculature, is it also possible to differentiate between other types of vasculature, to map them and to study the interactions between them? Is this tool limited to the skull, or can it be applied to other organs as well?

References

Ntziachristos V, Going deeper than microscopy: the optical imaging frontier in biology, Nat Methods, 2010, 7(8):603-614. doi: 10.1038/nmeth.1483.
Estrada H, Rebling J, Sievert W, Hladik D, Hofmann U, Gottschalk S,Tapio S, Multhoff G, Razansky D, Intravital optoacoustic ultrasound bio-microscopy reveals radiation-inhibited skull angiogenesis, bioRxiv, 2019, doi: https://doi.org/10.1101/500017

Tags: bone marrow, optoacoustic imaging, skull, vascular biology

doi: https://doi.org/10.1242/prelights.10644

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(2 votes)

1 comment

5 years

Hector Estrada

Dear Mariana,
Thank you for your interest in our work.
Answering your open questions:

1.- Given the non-invasive nature of the method, and since you mention previous links to radiation-induced long-term cognitive disability, would it be possible in the future, to combine this method with other imaging platforms such as functional MRI, to study the direct link of angiogenesis inhibition, with collateral effects of irradiation?

At the beginning of our study we knew that imaging bone vasculature in-vivo was challenging. We learned that it required at least two complementary techniques (optoacoustics and ultrasound) to tackle the problem. Then, in order to answer more involved biological questions such as how angiongenesis inhibition is linked to other collateral effects of irradiation, we certainly need to include other imaging platforms that could offer complementary information. We think it is technically feasible.

2.- In the past, your lab has generated hybrid systems combining fluorescent and optoacoustic methods. Could you combine your findings at mesoscopic level with fluorescence methods to image hematopoietic niche changes, and immune cell dynamics derived from damage to the calvarian microvasclature after irradiation?

In the current article we show what can be done with intrinsic contrast. Combining it with fluorescence imaging and sub-cellular resolution would result in a very exciting platform. Such a multiscale system can rely on optoacoustic and ultrasound to observe large scale effects of radiation and then zoom-in at an interesting spot to look at the details of cell dynamics using fluorescence labels. We also hope that people developing optoacoustic labels could soon help us reaching even further.

3.- Your study focused on the effects of irradiation on vascular remodelling, and you discuss effects in the context of cancer treatment. Your hybrid imaging method to visualize large areas of the skull, and to differentiate between cerebral and calvarian bone marrow could be applied to study other pathologies where changes to both compartments are induced. Have you considered applying this to other research topics?

We are certainly looking forward to using our method to study other pathologies that could affect the skull/brain interface. One could think of tumors and some other bone lesions.

4.- Given the possibility to differentiate calvarian from cerebral vasculature, is it also possible to differentiate between other types of vasculature, to map them and to study the interactions between them? Is this tool limited to the skull, or can it be applied to other organs as well?

It would be great if we could observe the lymphatic system as well. In order to do that we need either an extrinsic contrast or combine our method with fluorescence imaging.
Imaging bone vasculature in-vivo is technically very challenging, not only for optical methods. Ultrasound waves are greatly distorted when propagating through bone; in fact, most of the wave is reflected back. High resolution optoacoustic systems developed to image the mouse brain can only image very young mice or need to perform a craniotomy. Our method extends the applicability of high resolution optoacoustics and shows how to handle the skull and its vasculature properly. In addition, the skull is an excellent window to observe what happens inside the bone and it is right next to the brain, which is the most interesting organ. There are some examples in the literature that show segmentation of optoacoustic images in bone-less regions of the body. In our opinion, the skull is pivotal because it allows us to observe interesting biological processes and also paves the way for exploring other regions of the body where bones pose a problem for imaging.

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