1732834856681

OCT Monitoring of Biological Responses to
X-Ray, Photodynamic and Sonodynamic

Therapies in Rat Livers
Luismar Barbosa da Cruz Junior,1,* Iago Silva e Carvalho,2 Deise Luciane Paiva, 1
Mirian Denise Stringasci,1 Vanderlei Salvador Bagnato,1,2 and Sebastião
Pratavieira1
1São Carlos Institute of Physics, University of São Paulo, Av. Trabalhador São Carlense, 400, São Carlos,
13566-590, Brazil
2 Department of Biomedical Engineering, Texas AM University, 3120 TAMU, College Station, TX 77843, USA
* luismar@alumni.usp.br
Abstract: Monitoring photodynamic and sonodynamic therapies, along with ionizing

radiation, is important for assessing treatment efficacy. This study highlights the use of
OCT for evaluating the biological effects in rat livers. © 2024 The Author(s)
1. Introduction
Photodynamic and sonodynamic therapies are being widely implemented in various preclinical procedures com-
pared to traditional radiotherapy methods. To assess the efficiency of these techniques, animal studies are con-
ducted to evaluate their potential applications. In this context, the evaluation of the biological response to the
interaction of visible radiation, X-rays, and ultrasound waves becomes necessary, and OCT could be a potential
method for this purpose.
OCT is an optical technique based on the process of interferometry. It offers high resolution compared to other
optical techniques and has the advantage of being indirect, non-contact, non-invasive, and capable of obtaining
information about the morphological and anatomical structures of biological tissues [1]. In this study, we present
a structural and morphological evaluation of rat livers subjected to the three types of therapies and monitored the
biological responses using OCT.
2. Methodology
2.1. Treatment Protocols
All measurements were conducted in 3 Wistar Hannover Rats aged between 6 to 8 weeks, with weight ranging
between 200 grams up to 400 grams, and the irradiation was targeting the liver directly in all procedures.
Photodynamic Therapy: A 630 nm diode laser (Ceralax®, Ceramoptec, Germany) was used for 1000 s with a
power of 0.4 W over an area of 1 cm2 , over a distance of 2.1 cm from the source and the liver. The photosen-
sitizer Photogem® with 1.5 mg/kg was administered 30 minutes prior to the procedure. Euthanasia and OCT
measurements were performed 30 hours after PDT.
Sonodynamic Therapy: The procedure was performed using a Sonidel SP300 ultrasound device (Sonidel, Ireland),
delivering an intensity of 5W/cm2 with an transducer of effective radiation area 5.0 cm2 attached with a aluminum
cone as wave Guide during 5 minutes. The treatment was conducted in combination with Protoporfirin-IX (PpIX).
Euthanasia was carried out 30 hours following the sonication.
Radiotherapy: A dose of 5 Gy was applied to the center of the liver lobe using a XRAD 225x (Precision X-ray
Inc., USA) X-Ray source , with 40 cm distance of the radiation source. No sensitizers were used. Euthanasia was
performed 120 hours after the treatment.
2.2. Optical Coherence Tomography

OCT measurements were performed using the TELESTO-II SD-OCT (Thorlabs, Inc., USA), with a central wave-
length on 1300 nm, 76 kHz scanning rate, and pixel resolution of 5.5 µm. A refractive index of 1.38 was applied,
corresponding to the optical properties of rat liver tissue. The procedures were performed under controlled condi-
tions and temperature.
3. Results and Discussion

Figure 1 shows the OCT images of ex-vivo rat livers in response to PDT, X-ray, and SDT procedures, along with
a a healthy liver and a adhesive tissue structures.
Fig. 1: OCT images and its biological responses to (A) PDT (B) Radiotherapy, and (C) SDT. For comparison, a
(D) healthy liver and an (E) adhesive tissue of abdominal cavity viscera is shown.
In Figure 1 - (D) a healthy rat liver is shown. The OCT image shows an homogeneous medium with a visi-
ble Glisson’s capsule covering all the surface. The rat liver, like mammals in general, is primarily composed of
hepatocytes and is enveloped by Glisson’s capsule, a thin connective tissue layer that surrounds and protects the
hepatic structures.
The PDT response shown in Figure 1 - (A) does not exhibit significant morphological changes, however, it is
possible to observe a contrast difference in the tissue between the healthy and the damage region. The Glisson’s
capsule does not undergo significant alterations in this procedure. Liver tissues subjected to PDT exhibit color
changes in the irradiated region, transitioning from their characteristic dark coloration to a lighter tone [2]. On
the other hand, the radiotherapy shown in Figure 1 - (B), does not have a significant visual chance, however, its
seen on the OCT images an apparent second tissue layer on the liver surface, which is not observed in a healthy
liver. The second layer observed in the image may indicate the region where the tissue damage is restricted. The
Glisson’s capsule remains intact.
SDT uses mechanical waves to achieve its therapeutic effects. In Figure 1 - (C) is possible to see its effect on the
tissue, with a potential Glissom’s capsule rupture. Additionally, a second layer is also observed, but the damage
is irregular and deeper [2] copared with radiotherapy-treated subject. Lastly, Figure 1 - (E) shows the adhesive
tissue of abdominal cavity viscera, which is a fibrous connective tissue that forms as a result of an inflammatory
or scarring process. It is characterized by uniting or adhering surfaces of organs that are normally separated by
spaces lined with the peritoneum. It have its characteristic granular-shaped tissue and it may happens after some
abdominal surgery [3].
No histological slides were performed to confirm the damage area, but visual verification corroborates with
the OCT results. All animals subjected to the same procedures exhibited similar structural changes, indicating
consistent stimulation across each procedure. Previous study demonstrate a good correlation between histological
slides and OCT images [4], demonstrating the OCT potential use as a damage evaluation method in live animals
and real-time monitoring.
4. Conclusions
The SD-OCT system centered at 1300 nm is capable of identifying broader features, including general tissue
organization and macroscopic morphological changes induced by each therapy. The damage correlation with the
OCT images should be further investigated using histological slides for better understanding of damage formation.
References
1. S. Aumann, S. Donner, J. Fischer, F. Müller, “High Resolution Imaging in Microscopy and Ophthalmology: New
Frontiers in Biomedical Optics,” in New Frontiers in Biomedical Optics (Springer, 2019), pp. 1–407.
2. E. T. P. Ayala, F. A. D. de Sousa, J. D. Vollet-Filho, M. R. Garcia, L. De Boni, V. S. Bagnato, S. Pratavieira, “Pho-
todynamic and sonodynamic therapy with protoporphyrin IX: in vitro and in vivo studies,” Ultrasound in Medicine &
Biology, 47(4), 1032–1044 (2021).
3. D. Moris, J. Chakedis, A. A. Rahnemai-Azar, A. Wilson, M. M. Hennessy, A. Athanasiou, E. W. Beal, C. Argyrou,
E. Felekouras, T. M. Pawlik, “Postoperative abdominal adhesions: clinical significance and advances in prevention and
management,” Journal of Gastrointestinal Surgery, 21, 1713–1722 (2017).
4. Y. Winetraub, A. Van Vleck, E. Yuan, I. Terem, J. Zhao, C. Yu, W. Chan, H. Do, S. Shevidi, M. Mao, et al., “Noninvasive
virtual biopsy using micro-registered optical coherence tomography (OCT) in human subjects,” Science Advances,
10(15), 1–8 (2024).

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OCT Monitoring of Biological Responses to

X-Ray, Photodynamic and Sonodynamic

Abstract: Monitoring photodynamic and sonodynamic therapies, along with ionizing

2.2. Optical Coherence Tomography

3. Results and Discussion

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