Biology Imaging Hypoxia: Techniques, Reagents, and Imaging Methods

This report provides a comprehensive overview of hypoxia imaging, a critical medical condition affecting oxygen supply at the tissue level. It explores the reasons for imaging hypoxia, emphasizing its impact on tumour treatment outcomes and the development of molecular imaging techniques. The report details various methods for imaging hypoxia, including PET, NIR, MRI, and EPR, and discusses the evolution of hypoxia markers like 2-Nitroimidazole compounds. A significant portion of the report is dedicated to the reagents used in hypoxia imaging, such as Image-It Red and Green hypoxia reagents, Pimonidazole, and FMISO, explaining their mechanisms and applications. It also delves into reagent design, highlighting the role of targeting vectors and the properties required for effective tracers. Finally, it examines the imaging techniques, including radionuclide detection and PET, used to visualize and analyze hypoxia in biological systems.

Running head: BIOLOGY
IMAGING HYPOXIA
Name of the Student
Name of the University
Author Note

Running head: BIOLOGY
Table of Contents
Introduction................................................................................................................................3
Hypoxia..................................................................................................................................3
Reason of imaging hypoxia....................................................................................................3
Methods for imaging hypoxia................................................................................................3
Body...........................................................................................................................................4
Reagents used in imaging hypoxia.........................................................................................4
Reagent design.......................................................................................................................6
Targeting vectors....................................................................................................................7
Imaging..................................................................................................................................8
Conclusion..................................................................................................................................9
References................................................................................................................................11

Running head: BIOLOGY
Introduction
Hypoxia
Hypoxia is a medical condition which has been found to affect a specific organ or a region of
the human body that lacks an adequate supply of oxygen at the tissue level. This is a general
pathological condition of the human body that affects the region of the body. Hypoxia has
often been found as a pathological disorder which varies with the changes in arterial
concentrations. This can occur during normal physiology or during hypoventilation training,
for example, physical exercise (Eales, Hollinshead and Tennant 2016). Hypoxia has been
found to differ from anoxemia and hypoxemia which refers to the oxygen supply state and is
insufficient. Hypoxia associated with full deprivation of oxygen supply is called anoxia.
Reason of imaging hypoxia
A hypoxic condition has the consequences where the patient with hypoxic tumours
experiences poor outcomes for every treatment. This occurs because when cells are exposed
to hypoxic conditions for long, they start secreting proteins which are required for their
survival. Thus, the cells grow spontaneously in the hypoxic conditions giving rise to tumours
and a metastatic condition This condition has led to the development of molecular imaging
techniques of hypoxia in cells (Hirata et al. 2019). Some of the techniques include regional
measurements from oxygen electrodes which are placed under the guidance of CT and MRI
(magnetic resonance imaging). Hypoxia imaging has become a great success story since the
past decades and also provides a model for significant lessons in molecular imaging
techniques or probes.
Methods for imaging hypoxia
The methods for measuring or imaging hypoxia can also be termed as the methods for
imaging the condition corresponding to tissue oxygenation. The following techniques have

Running head: BIOLOGY
been used the most for measuring tissue hypoxia- PET (positron emission tomography), near-
infrared or NIR, phosphorescence, MRI or magnetic resonance imaging and BOLD MRI
including electron paramagnetic resonance of EPR. 2-Nitroimidazole compounds have been
originally derived for hypoxic cells to act as radiosensitisers and were introduced as the
hypoxia markers in the year 1970 (Klockow, Hettie and Chin 2018). These compounds have
been stated to enter the cells by the process of passive diffusion in which they can start
undergoing the reduction process which forms a reactive intermediate species. Accurate
quantification of tissue oxygenation and the variation in the ability to quantify the same has
been found to be the differentiating factors for the techniques stated in the previous line. This
paper will discuss the core models for imaging hypoxia and the reagents used for performing
the same process.
Body
Reagents used in imaging hypoxia
Reagents are specific chemic compounds which are used to perform or visualize a chemical
reaction. Reagents can either be fluorogenic or non-fluorogenic. Some of the reagents can
have a dual function of becoming a fluor after the binding of a specific chemical group.
Fluorogenic reagents have been used for the imaging process from a very long time in
medical science (Huang et al. 2019). Fluorescence technology has been used in biophysical
chemistry in order to study the progression of various chemical reactions and identification of
a specific substance of interest inside the cells (Geng et al. 2019). Microscopes based on the
technology of fluorescence has been discovered for the detection of microorganisms along
with their produced materials. Thus, it can be stated that reagents will also be required to
image the process of hypoxia although it is not a chemical reaction (Davis and Schneider et
al. 2018). Hypoxia has been stated to be a medical condition that has been found to affect a
specific organ or a region of the human body that lacks an adequate supply of oxygen at the

Running head: BIOLOGY
tissue level. This is a general pathological condition of the human body that affects the region
of the body. Hypoxia has often been found as a pathological disorder which varies with the
changes in arterial concentrations. Most of the reagents used in hypoxia imaging are fluors
and react to variations in the environment depending on the availability of oxygen.
Starting the discussion for reagents used to image hypoxia, Image-It Red hypoxia reagent has
been identified as the novel fluorogenic compound which is used for the measurement of
hypoxia that is occurring in living cells (Papkovsky and Dmitriev 2018). This reagent is non-
fluorescent when live cells are present inside an environment associated with normal cells
with oxygen concentration and have been found to become fluorescent when oxygen levels
have started decreasing. This reagent has been considered as a real-time detector of oxygen
and has been found to be associated with the fluorogenic response which reverses when the
cells are restored back to their normal state. This property of red reagent has been increasing
its importance in the field of imaging hypoxia in human tumour cells. When the surrounding
oxygen levels are near 5%, this reagent has been designed to fluoresce, signifying a hypoxic
condition in the selected cells.
Next comes the green hypoxia reagent which is also a unique and fixable fluorogenic
compound which has been used for the measurement of hypoxia inside living cells. Since it is
very much non-fluorescent in regions where living cells are in a normal oxygen environment,
it is of more use than red reagent. This is because of the fact that this reagent produces green
colour with less wavelength at low oxygen concentration levels and thus can be used to
sustain the signal when the tissues start returning to their normal levels of oxygen (Yu et al.
2017). This reagent is stated to be permeable to living cells and become fluorescent inside the
low oxygen environments. Pimonidazole has been stated to be another similar reagent which
only responds to very low oxygen concentrations and thus can be said as less sensitive than
the green reagent.

Running head: BIOLOGY
2- Nitroimidazole based reagents have been broadly used in nuclear and pathological
medicine examination and tests for a long time (Ruan et al. 2019). These exams and tests are
mainly associated with the detection of hypoxic areas inside tumours. Pimonidazole has been
used for the process of histochemical staining associated with hypoxic areas. This reagent has
been found to accumulate inside the hypoxic cells through covalent bonding with the
macromolecules which gives rise to reductive metabolites after the complete reduction of the
nitro group. In LC-MS analysis, a glutathione conjugate of reduced pimonidazole has been
found to be widely used in the imaging process of hypoxic tumour cells.
FMISO or the 18F- fluoromisonidazole has been used to the accumulation of hypoxic tumour
tissues which uses IMS combined with the process of radiographic analysis (Liu, Bu and Shi
2017). This process has been widely used in the imaging of hypoxia with probes for the
diagnosis of PET (Zhang et al. 2019). These are the overall reagents used in hypoxia, all of
which has similar mechanisms of action.
Reagent design
There are three main methods for designing the reagents to be used for hypoxia imaging. This
process is also known as the probe design procedure which states that targeting vector linked
bimodal probes have been used in the imaging process. The nitroimidazole family of
compounds have been used in a large number of tracers for the identification of hypoxia in
solid tissues and tumours. The main driving force behind the design has been found to be the
need for a unique imaging procedure in order to get rid of the inconsistent associations
between imaging and finding modalities which include PET. Medical image modalities have
been found to include positron emission tomography or PET which is associated with SPECT
and hybrid imaging systems. Modality has been defined as a term which has been used in
radiology in order to refer a single form of imaging which includes CT scanning. The
molecules designed as the reagents have been stated to be called as reporter molecules and

Running head: BIOLOGY
are safe for human cells at a very low concentration. Various endogenous molecular markers
have been designed and studied for cervical cancer patients. HIF-1 is one of the most reliable
markers for tumour hypoxia. GLUT-1 is another endogenous hypoxia marker for hypoxia in
oral, squamous cell and rectal carcinomas. Carbonic anhydrase or CA-9 marker has been
used as a prognostic indicator for cervical cancer. These markers are so designed that they
exhibit a number of specific chemical properties. The tracer has been found to readily enter a
cell and that too in a non0specific manner. This compound is also designed in such a way,
that it leaves the cell when the oxygen concentrations become normal. Reagents designed will
be retained in regions with hypoxia and will be released in regions with a high partial
pressure of oxygen. The markers are designed to be lipophilic so that they enter their target
cells and finally allow their distribution throughout the tissue in a uniform pattern. The
marker should not be destroyed by non-hypoxic metabolism. The reagent should be designed
such that it is repeatable to allow both the return to normoxia and detection of hypoxia. All
the tracer compounds must have the procession of getting easily removed with a high
clearance rate from blood after the multimodal imaging process has been completed.
Targeting vectors
Targeting vectors are the compounds which are targeted in order to give rise to the reagents
to be used for the imaging purpose.
2-Nitroimidazole compounds have been originally derived for hypoxic cells to act as
radiosensitisers and were introduced as the hypoxia markers in the year 1970. These
compounds have been stated to enter the cells by the process of passive diffusion in which
they can start undergoing the reduction process which forms a reactive intermediate species.
During normoxic conditions, The reduction of nitroimidazoles has been found to require the
presence of active tissues including reductases which are detected by this compound.

Running head: BIOLOGY
18F-fluoromisonidazole has been found to act as one of the major sources of fluorinated
nitroimidazole based markers which were synthesized for PET imaging process. This
compound has been found to constitute a prototype 2 nitroimidazole tracer and has been
mostly used as the most significant PET hypoxia marker (Kumar et al. 2017). This compound
has been stated to consist of hypoxia determining ability because it fluoresces in changes with
the surrounding oxygen. Due to the ability of hypoxic selectivity of FIMSO, it can be stated
that it leads the candidate assessment of hypoxia along with PET (Xie et al. 2016). However,
it has been observed that FIMSO has a slow pharmacokinetic profile with a very limiter
clearance profile.
One of the most significant quinolones and metal combined reagent is Cu-diacetyl-bis(N4-
methylthiosemicarbazone) which has been used as the alternative class of reagents for
hypoxia study. Cu-ATSM has been used as a prototype molecule because it has low
molecular weight and lipophilicity. Thus, it can be stated that this compound has high
membrane permeability which is one of the primary characteristics for an ideal tracer for
hypoxia. Under hypoxic exposure, this compound has been found to be unstable and
spontaneously dissociate into copper and ATSM which leads to the process of intracellular
Cu ion trapping (Fan et al. 2020). However, in the presence of oxygen, the reverse process
occurs which leads to the re-oxidation of the dissociated ions to the parent compound. Thus,
it can be stated that this compound is also known as the oxidation reduction process and the
compound can be categorized as the redox active metal complexes.
Imaging
Radionuclide detection has been found to be associated with tumour detection with the use of
14-C misonidazole autoradiography (Gérard et al. 2019). The process of PET for imaging
hypoxia has been found to be associated with two main classes of tracer which has been
developed in order to study specifically hypoxia. 18-F labelled nitroimidazole molecules and

Running head: BIOLOGY
Cu-labelled bis(N4-methylthiosemicarbazone) analogues have been used the most for the
imaging process using PET. Various methods for imaging or measuring hypoxia can also be
termed as the methods for imaging the condition corresponding to tissue oxygenation. There
are several techniques that have been used the most for measuring tissue hypoxia- PET
(positron emission tomography), near-infrared or NIR, phosphorescence, MRI or magnetic
resonance imaging and BOLD MRI including electron paramagnetic resonance of EPR
(O'Connor, Robinson and Waterton 2019).

Running head: BIOLOGY
Fig 1: MRI hypoxia
Source: Knox et al. (2018)
Another compound known as 18F-3-fluoro-2-(4-((2-nitro-1H-imidazole-1-yl)methyl)-1H-
1,2,3-triazole-1-yl)propan-1-ol (18F-HX4) contains a 1,2,3-anti-triazole moiety (as a
synthetic convenience) that makes it more hydrophilic than its parent compound (FIMSO).

Running head: BIOLOGY
This compound has been used in imaging hypoxia associated with tumours of neck and head.
According to recent studies, RP-170 (1-(2-1-(1H-methyl)ethoxy)methyl-2-nitroimidazole),
another 2-nitroimidazole-based hypoxic radiosensitiser, has also been labelled with 18F to
use in hypoxia imaging. Pieces of research articles found that the imaging process of tumour
hypoxia with oxygen-enhanced MRI or the BOLD MRI has the potential to identify and map
the tumour hypoxia (Xu et al. 2017). MRI is a process used in radiology to scan a particular
region by using the principle of magnetism and produce the image of body regions. Scientists
have found that MRI can also be used to image hypoxia where the patient is placed inside a
large moveable bed which is inside a giant circular magnet. The spins of various protons
inside the cell during a hypoxic condition has been found to differ from the variation of
protons inside the cell during a normal condition. Thus, it can be stated that hypoxia can be
imaged by the use of MRI. Presently hypoxia imaging processes have been found to be
available for daily clinical use. The process uses changes un overall longitudinal relaxation or
the R1 and effective transverse relaxation or the R2 of the oxygenation level of blood which
is induced by 100% oxygen or the radiosensitising gas known as carbogen.

Running head: BIOLOGY
Fig 2: PET imaging of hypoxia
Source: Silvoniemi et al. (2018)
Conclusion
After the long discussion, it can be stated that Hypoxia is a medical condition which has been
found to affect a specific organ or a region of the human body that lacks an adequate supply
of oxygen at the tissue level. This condition is a general pathological situation of the human
body that affects the region of the body. Hypoxia has often been defined as a pathological
disorder which varies with the changes in arterial concentrations. Imaging processes have
shown that this condition can occur during normal physiology process or during
hypoventilation pieces of training, for example, physical exercise. This paper shows the
methods for measuring or imaging hypoxia can also be termed as the methods for imaging
the condition corresponding to tissue oxygenation. According to the paper techniques found
to have been used the most for measuring tissue hypoxia- PET (positron emission
tomography), near-infrared or NIR, phosphorescence, MRI or magnetic resonance imaging
and BOLD MRI including electron paramagnetic resonance of EPR. 2-Nitroimidazole
compounds have been originally derived for hypoxic cells to act as radiosensitisers and were
introduced as the hypoxia markers. These compounds have been stated to enter the cells by
the process of passive diffusion in which they can start undergoing the reduction process
which forms a reactive intermediate species. Accurate quantification of tissue oxygenation
and the variation in the ability to quantify the same. The condition of hypoxia has been found
to differ from anoxemia and hypoxemia which refers to the oxygen supply state and is
insufficient. Hypoxia associated with full deprivation of oxygen supply is called anoxia.
Thus, it can be stated that out of all the imaging process (non-invasive and invasive), PET
and MRI have been stated as the most useful ones from the list.

Running head: BIOLOGY

Running head: BIOLOGY
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Running head: BIOLOGY
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Running head: BIOLOGY
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