RU2804844C9 - Method for obtaining contrast-enhanced ct images of liver of small laboratory rodents during intravital radiation imaging to asses the presence and growth dynamics of malignant neoplasms - Google Patents

Abstract

FIELD: medicine; experimental oncology.

SUBSTANCE: invention can be used to conduct experimental intravital computed tomography (CT) of the liver of mice. The mouse is put in anesthetized sleep. Next, the mice are injected with an MR contrast agent based on gadoxetic acid “Primovist” in a volume of 500 mcL for 1.5 minutes. A CT scan of the mouse liver is performed. Using the obtained images of the liver, the X-ray density in the area of interest is determined and a “X-ray density – study time” curve is constructed. The resulting curve is compared with the calibration curve “X-ray density - study time”, which is constructed so that at time 0 min the X-ray density corresponds to the average X-ray density of the liver before the administration of Primovist; at 90 minutes the X-ray density reaches a maximum value of 220-235 HU, from 91 minutes to 160 minutes, the retention phase begins, in which the radiological density is 220-235 HU; from 161 minutes to the end of the study time, the elimination phase begins, in which the radiological density decreases.

EFFECT: method provides an assessment of the growth dynamics of malignant neoplasms by comparing the calibration and experimental curves “X-ray density - study time”.

1 cl, 5 dwg

Description

The invention relates to medicine, namely experimental oncology, and can be used for intravital radiation imaging of organs and tissues of model organisms of small laboratory rodents to study under experimental conditions the features of their anatomical systems with tumors of various localizations, clarify the parameters of their blood supply, detect metastatic foci and clarification of the anatomical and topographical features of the animal for further manipulations [Pandharipande PV, Krinsky GA, Rusinek H, Lee VS. Perfusion imaging of the liver: current challenges and future goals. Radiology. 2005;234(3):661-73. doi:10.1148/radiol.2343031362].

Radiation imaging methods provide a fairly clear picture of the state of internal organs and their systems in living small laboratory animals, whose clinical examination and/or the use of ultrasound diagnostics is difficult due to the size of the object being studied. However, there is difficulty in determining the contours of the abdominal organs, in particular the liver and spleen, since all currently existing angio-contrasts are nonspecific for these organs [Lauber DT, Fülöp A, Kovács T, Szigeti K, Máthé D, Szijártó A. State of the art in vivo imaging techniques for laboratory animals. Lab Anim. 2017;51(5):465-78. doi:10.1177/0023677217695852]. Methodological approaches have now been developed to approximately identify these anatomical structures individually. One of the most significant abdominal organs for contrasting with any of the modern available methods is the liver. To detect it and study anatomical structures in vivo, nanoparticles and/or liposomes/micelles/fluorescently labeled molecules or peptides are used - targeted targeting molecules labeled with a radioisotope substance [Topcu O, Kurt A, Nadir I, Arici S, Koyuncu A, Aydin C Effects of contrast media on the hepato-pancreato-biliary system. World J Gastroenterol. 2009;15(38):4788-93. doi:10.3748/wjg.15.4788; Cuenod C, Leconte I, Siauve N, Resten A, Dromain C, Poulet B, Frouin F, Clément O, Frija G. Early changes in liver perfusion caused by occult metastases in rats: detection with quantitative CT. Radiology. 2001;218(2):556-61. doi:10.1148/radiology.218.2.r01fel0556; Torchilin VP, Frank-Kamenetsky MD, Wolf GL. CT visualization of blood pool in rats by using long-circulating, iodine-containing micelles. Acad Radiol. 1999;6(l):61-5. doi: 10.1016/sl076-6332(99)80063-4; Ehling J, Theek B, Gremse F, Baetke S, Möckel D, Maynard J, Ricketts SA, Grüll H, Neeman M, Knuechel R, Lederle W, Kiessling F, Lammers T. Micro-CT imaging of tumor angiogenesis: quantitative measures describing micromorphology and vascularization. Am J Pathol. 2014; 184(2):431-41. doi:10.1016/j.ajpath.2013.10.014].

Modern technologies make it easier for the researcher to identify areas of interest. The only problem is the peculiarity of the physiology of bile secretion from the body of small laboratory animals. As soon as the volume of circulating fluid (water fraction of blood) increases, the viscosity of bile simultaneously decreases, and it, together with the administered contrast compound, fills the intestinal loops that are adjacent to the liver. 3-5 minutes after the introduction of classical CT contrasts, it is not possible to differentiate intestinal loops from the liver.

Currently, the introduction of radiocontrast agents into the body of laboratory rodents is carried out using the bolus technique [Ting Liu, Sheng Wang, Wei Zhao Hao Liu, Yi Zhang, Juan Han, Xia Chen. Experimental Analysis of the Utility of Liquid Metal Gallium as a Contrast Agent for CT Hepatic Artery Angiography in Living Rabbits, 30 December 2020, PREPRINT (Version 1) available at Research Square, doi: 10.21203/rs.3.rs-135998/v1; Ehling J, Theek B, Gremse F, Baetke S, Möckel D, Maynard J, Ricketts SA, Grüll H, Neeman M, Knuechel R, Lederle W, Kiessling F, Lammers T. Micro-CT imaging of tumor angiogenesis: quantitative measures describing micromorphology and vascularization. Am J Pathol. 2014; 184(2):431-41. doi: 10.1016/j.ajpath.2013.10.014; Starosolski Z, Villamizar CA, Rendon D, Paldino MJ, Milewicz DM, Ghaghada KB, Annapragada AV. Ultra High-Resolution In Vivo Computed Tomography Imaging of Mouse Cerebrovasculature Using a Long Circulating Blood Pool Contrast Agent. Sci Rep.2015;5:10178. doi: 10.1038/srep10178; Ghanavati S, Yu LX, Lerch JP, Sled JG. A perfusion procedure for imaging of the mouse cerebral vasculature by X-ray micro-CT. J Neurosci Methods. 2014;221:70-7. doi: 10.1016/j.jneumeth.2013.09.002; Hlushchuk R, Haberthür D, Soukup P, Barre SF, Khoma OZ, Schittny J, Haghayegh Jahromi N, Bouchet A, Engelhardt B, Djonov V. Innovative high-resolution microCT imaging of animal brain vasculature. Brain Struct Function. 2020;225(9):2885-95. doi:10.1007/s00429-020-02158-8]. Data from the literature available for analysis indicate that radiocontrast agents can be injected into the tail vein of an animal for a sufficiently long time and in high concentrations [Akladios CY, Bour G, Raykov Z, Mutter D, Marescaux J, Aprahamian M. Structural imaging of the pancreas in rat using micro -CT: application to a non-invasive longitudinal evaluation of pancreatic ductal carcinoma monitoring. J Cancer Res Ther. 2013; 1 (2):70-6. doi: 10.14312/2052-4994.2013-11; Toy R, Hayden E, Camann A, Berman Z, Vicente P, Tran E, Meyers J, Pansky J, Peiris PM, Wu H, Exner A, Wilson D, Ghaghada KB, Karathanasis E. Multimodal in vivo imaging exposes the voyage of nanoparticles in tumor microcirculation. ACS Nano. 2013;7(4):3118-29. doi: 10.1021/nn3053439; Clark DP, Badea CT. Micro-CT of rodents: state-of-the-art and future perspectives. Phys Med. 2014;30(6):619-34. doi: 10.1016/j.ejmp.2014.05.011; Lee YC, Fullerton G, Goins B. Comparison of multimodality image-based volumes in preclinical tumor models using In-Air micro-CT image volume as reference tumor volume. Open Journal of Medical Imaging. 2015;5(03):117-32. doi: 10.4236/ojmi.2015.53016].

This method has a drawback - the distribution of the contrast compound occurs almost from the first second of administration, and the quality of the resulting images may not be sufficient due to its rapid removal from the animal’s body. Therefore, it is necessary to administer a large amount of the compound at once, which can provoke toxic damage to the excretory organs, primarily the kidneys.

There are registered hepatospecific contrast agents that are intended exclusively for MRI imaging (for example, “Primovist”) https://grls.rosminzdrav.ru/Grls_View_v2.aspx?routingGuid=533a3325-f30f-4d7b-9132-c5638371dd7f). They can be adapted for use in experimental animals for the purpose of CT studies, since the active substance of the drug substance is a radiodense compound. If this method is to be used, then the animal must be constantly injected with a highly diluted radiocontrast agent intravenously during the examination directly in the CT machine at a very low speed. This method is not optimal.

It is possible to use another option: a concentrated official drug (for example, “Primovist”) is administered to the animal in the maximum permissible dose by weight, after which the dynamics of its accumulation in the organ of interest is determined, a calibration curve is constructed taking into account standard time points, and studies of animals of the same type by weight are carried out [ Döhr O, Hofmeister R, Treher M, Schweinfurth N. Preclinical safety evaluation of Gd-EOB-DTPA (Primovist). Invest Radiol. 2007;42(12):830-41. doi: 10.1097/RLI.0b013e318137a471; Schmitz SA, Wagner S, Schuhmann-Giampieri G, Krause W, Wolf KJ. A prototype liver-specific contrast medium for CT: preclinical evaluation of gadoxetic acid disodium, or Gd-EOB-DTPA. Radiology. 1997;202(2):407-12. doi:10.1148/radiology.202.2.9015066]. This option does not provide the researcher, when conducting modern CT studies, with the opportunity to trace the dynamics of drug distribution in one animal over a long period of observation, since the necessary and sufficient amount of the drug during anesthesia is not excreted at the required speed and damages the kidneys.

The use of a hepatospecific drug for MP contrast based on gadoxetic acid - “Primovist” in humans [https://mrtsurgut.ru/wp-content/uploads/2017/02/Primovist-slide-kit-l-1.pdf] by us was accepted as a prototype.

Objective of the invention:

adaptation of the use of a hepatospecific drug for MP contrast based on gadoxetic acid in small laboratory rodents, providing X-ray contrast of their liver for experimental studies.

The problem is solved by creating a new method for obtaining contrast-enhanced CT images of the liver of small laboratory rodents during intravital radiation imaging to assess the presence and growth dynamics of malignant tumors based on the use of calibration phantoms. The method involves initiating anesthesia in experimental animals, providing access to a peripheral vein in which an intravenous cannula/injection needle is installed, and ensuring the uninterrupted flow of the drug into the bloodstream. Upon completion of this stage, the test animal is introduced into the gantry of a computed tomograph, then the finished dosage form is administered, a hepatospecific X-ray contrast agent for MR contrast based on gadoxetic acid with a concentration of 181.430 mg/ml in the maximum tolerated dose with a single administration to mice for 1.5 minutes, 5.5 μl/sec or 0.33 ml/min and scan the area of interest, including the possibility of dynamic scanning. The resulting CT images of the liver are compared with a calibration curve, which is obtained by successively diluting the contrast agent in microtubes of a phantom model and then shooting them in a CT machine. If the dynamics of distribution and retention of the contrast agent in the organ differ significantly from the calibration curve, then it is necessary to analyze the presence of malignant neoplasms.

Preliminary experimental data led to the conclusion that the introduction of a hepatospecific contrast agent, which is intended for MP imaging, can be used for experimental imaging of the mouse liver during CT scanning. To check the quality of visualization and to evaluate the possibility of using a hepatospecific MP contrast agent, a phantom was used, which consisted of microtubes with contents having different radiological properties and simulating an isolated organ in which the accumulation and/or retention of hepatospecific contrast occurred.

Figure 1a shows the arrangement of microtubes: A - microtube with air, B - microtube with distilled water, C - microtube with "Primovist" 0.25 M, which were taken in optical black and white photography mode.

Figure 1b shows CT images of the same microtubes. Dynamic range of the image: -1500÷2000 HU. A - microtube with air - 1000±4 HU; B - microtube with distilled water - 2±3 HU; B - microtube with “Primovist” 0.25 M for active substance - 1743 ± 6 HU). Thus, typical 3 variants of CT images were obtained, differing in radiodensity value: (1) air (the radiodensity value was taken as 0 in this study); (2) contrast at maximum concentration (the radiodensity value is taken as 1 in this study) without dilution; (3) water (including any liquid without a contrast agent, may have a value of 1). If necessary, using the conditional straight line “air-drug” or “air-water” or “drug-liquid”, you can carry out serial measurements of the resulting X-ray densities in microtubes and, using their data, calibrate the device so that its performance characteristics meet the requirements of the study.

In this case, the following scanning parameters were chosen as optimal:

X-ray tube voltage - 50 kV;

X-ray tube current - 0.21 mA;

Projection exposure time - 75 ms;

X-ray tube pitch angle - 0.5°;

X-ray tube filter - Al 100+400 microns.

Reconstruction and image processing parameters:

Voxel size - 60 µm;

Image filter - Gaussian Smooth 0.18 mm.

Using the instrument's built-in software, based on standard CT image reconstruction methods, volumetric linear X-ray absorption coefficient maps (standard CT images) were obtained.

Thus, it was decided to conduct a study to obtain images of the liver of laboratory rodents (mice, rats) using computed tomography in the first 3-5 minutes of scanning with a constant supply of a hepatospecific radiocontrast agent through a controlled duct system (it is possible to obtain the required degree of accumulation in the organ with constant monitoring minute by minute).

Technical result. Our proposed method for monitoring the rate of entry of a contrast agent into the animal’s body allows the researcher to assess the accumulation of a contrast agent in areas of interest (liver), determine the dynamics of its entry into the organ parenchyma and/or tumor, determine the localization, degree and prevalence of pathological changes in the liver in that case , if necessary. The difference from the prototype is the ability to control the rate of entry of the drug into the body of the animal being studied, which allows us to examine in detail the main areas of the liver parenchyma that have retained their physiological functions.

The inventive method is illustrated by the following figures:

In fig. 1 - black and white photograph (a) and CT image (b) of microtubes that form the basis of the phantom model, which contain air, water and X-ray contrast “Primovist” 0.25 M for contrasting the liver.

In fig. 2 - computed tomogram of the mouse body: a - coronal projection before contrast injection; b - coronal projection after contrast injection, the area of interest - the liver - is highlighted with a rectangle.

In fig. 3 - computed tomogram of the mouse body: a - sagittal projection before contrast injection; b - sagittal projection after contrast injection, the area of interest - the liver - is highlighted with a rectangle.

In fig. 4 - dynamics of changes in the radiodensity of mouse liver parenchyma after a single injection of a contrast drug. The radiodensity at time t=0 min on the graph corresponds to the average radiodensity of the liver before the administration of Primovist. The error in the average X-ray density is due to instrument error and the heterogeneity of the volume under study.

The method is carried out as follows. Anesthesia sleep is initiated in experimental animals, access to a peripheral vein is provided, in which an intravenous cannula/injection needle is installed, and the uninterrupted flow of the drug into the bloodstream is ensured. Upon completion of this stage, the animal under study is introduced into the gantry of a computed tomograph.

Next, the finished dosage form is administered - a hepatospecific X-ray contrast agent for MR contrast based on gadoxetic acid with a concentration of 181.430 mg/ml in a volume of 500 μl (the maximum tolerated dose for a single administration to mice) [Döhr O, Hofmeister R, Treher M, Schweinfurth N. Preclinical safety evaluation of Gd-EOB-DTPA (Primovist). Invest Radiol. 2007;42(12):830-41. doi: 10.1097/RLI.0b013e318137a471; Schmitz SA, Wagner S, Schuhmann-Giampieri G, Krause W, Wolf KJ. A prototype liver-specific contrast medium for CT: preclinical evaluation of gadoxetic acid disodium, or Gd-EOB-DTPA. Radiology. 1997;202(2):407-l2. doi: 10.1148/radiology.202.2.9015066] for 1.5 minutes (5.5 μl/sec or 0.33 ml/min) and scan the area of interest, including the possibility of dynamic scanning. The scan was performed under the following conditions: computed tomography (CT) in a MiLabs VECTor6 tomograph (Netherlands). Post-processing and analysis of images is carried out in the PMod program.

The result of the obtained tomograms is figures 2-3;

Dynamic characteristics - figure 4.

The basis for the proposed method is the results of experimental studies on phantom models and performed on mice weighing 20-25 g. After obtaining a CT image of a hepatospecific contrast agent and calculations possible for administering doses of the drug, the mice were selected and placed in a box for inhalation delivery isoflurane. Next, access to a peripheral vein was provided for the administration of a contrast agent, after which the animal was placed in a specialized holding device - a crib - for computed tomography with a constant flow of isoflurane and scanning began.

Thus, a method for obtaining images of the liver of mice during intravital radiation imaging was developed and tested, including for assessing the presence and growth dynamics of malignant tumors. This approach, used for the needs of experimental oncology, allowed us to solve the following problems:

(1) identify and delineate the liver, accurately visualizing the boundaries of the organ from the adjacent spleen and intestinal loops (Figs. 2 and 3);

(2) establish the dynamics of accumulation of an MP contrast agent based on gadoxetic acid with a concentration of 181.430 mg/ml when administered in the maximum volumes allowed for mice of a given weight (Fig. 4);

(3) obtain an image of the entire liver and determine the time of maximum accumulation of the drug in the organ - from the fortieth to the ninetieth minute of observation, which corresponded to a radiodensity of 220-235 HU (Fig. 2-4).

The radiodensity of the liver parenchyma (Fig. 4) at time 0 min corresponds to the average radiodensity of the liver before the administration of Primovist. Over time, the drug accumulates (from 0 min to 90 min), followed by a retention phase (from 91 min to 160 min), followed by elimination (from 161 min to the end of observation). Thus, if necessary, it is possible to obtain dynamic characteristics of the movement of the drug through the working tissue of the liver, focusing on the data presented in Fig. 4. If the dynamics of distribution and retention of the drug in the organ in any way significantly differs from a calibration curve of this type, then it is worth saying that the parenchyma has a heterogeneous structure and it is necessary to analyze the presence of malignant neoplasms.

This method makes it possible to obtain data without removing animals from experimental work, which is important for developing methods of experimental oncology, namely when modeling organotropic malignant neoplasms, their metastases and, in particular, primary liver tumors.

Claims (1)

A method for conducting experimental intravital computed tomography (CT) tomography of the liver of mice, which consists in initiating anesthetized sleep in the mouse, then injecting the mouse with an MR contrast agent based on gadoxetic acid “Primovist” in a volume of 500 μl for 1.5 minutes and performing a CT scan mouse liver, on the obtained images of the liver, determine the X-ray density in the area of interest and construct a curve “X-ray density - study time”, compare the resulting curve with the calibration curve “X-ray density - study time”, which is constructed so that at time 0 min the X-ray density corresponds to the average radiological density of the liver before the introduction of Primovist, at 90 minutes the radiological density reaches a maximum value of 220-235 HU, from 91 minutes to 160 minutes the retention phase begins, in which the radiological density is 220-235 HU, from 161 minutes to the end of time The study begins the elimination phase, during which the radiological density decreases.