Methods

Oxygen-Sensitive Paramagnetic Particle, Lithium Phthalocyanine

3. Methods

3.1. Process Outline In vivo EPR oxymetry experiment typically includes the following steps:

1. Selection of the target organ to measure pO2; 2. Establishing the standard pO2calibration curve;

3. Implantation of LiPc particle in one or several target organs;

4. Stabilization for at least 4–7 d prior to starting the measure-ments;

5. Measurement of pO₂in the target organ using EPR; and 6. Data processing (conversion of line width (mGauss) to pO₂

(mmHg) using the calibration curve).

3.2. pO₂Calibration (Standard ) Curve

In order to obtain the in vivo pO₂data from the conversion of the acquired EPR line width of LiPc to the corresponding pO2level, the establishment of pO₂calibration (standard) curve is required.

An aliquot (1–3 mg) of LiPc particles suspended in PBS (10–

20␮L) is taken in a gas-permeable Teflon tube. The Teflon tube is placed on a surface coil type resonator with the LiPc particles at the center of the coil (Fig. 2.3). The resonator and sample are covered with a plastic tube and warmed to 36–37^◦C using a combination of hot air and an IR lamp (see Note 3).

The EPR line widths of the LiPc particles are measured under several conditions of gas flow, including 21 (medical air), 15, 10, 5, 1.25 (O₂/N₂), and 0% (argon), and then a calibration curve for LiPc oxymetry is obtained. The measurements are done after

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Gas flow 700 mL/min

700 MHz surface coil (i.d. 7.3 mm) Gas permeable tubing filled with PBS

Plastic tubing

IR lamp

LiPc crystal Warmed air

Non-magnetic temperature probe

Fig. 2.3. Schematic representation of the LiPc setting for pO₂ calibration (standard) curve.See Section 3.2 and Note 3 for additional information on the placement of Teflon tube, surface coil type resonator, and the LiPc particles.

at least 30 min equilibration under each gas flow condition at the rate of 700 mL/min. The EPR signal is measured by CW EPR at 700 MHz using a single loop surface coil resonator.

3.3. Implantation of LiPc Particles

For the in vivo EPR oxymetry with oxygen-sensitive probe, the LiPc particles can be implanted in several target organs. As the surface coil is placed on one preferred site of target organ/tissue, we can repeatedly measure the pO₂ level in a specific/localized area (see Note 4).

The mouse is anesthetized by administering 1.5% isoflurane in medical air (flow rate was 700 mL/min) through a nose cone.

The fur of lower abdomen and femoral region is removed by shav-ing. A portion of LiPc particles (5–10 mg) is suspended with an appropriate volume (10–20␮L) of physiologic saline and kneaded until a slurry paste is obtained. Then, the LiPc slurry is placed in the tip of a 20-gauge injection needle to make a soft pellet 2–3 mm long. The needle is injected into the desired region of the animal, and the pellet is pushed out using a smooth-fitting piston.

Fig. 2.4shows a diagram of the LiPc implantation scheme. The average dry weight of the pellet is 0.8± 0.1 mg (mean ± SD), which is estimated from 7 randomly selected pilot runs. For exam-ple, implantations of LiPc are made into both the right femoral muscle and the left lower abdominal mammary gland pad of the mouse (see Note 5) The quantitation of pO₂ is achieved by the calibration curve shown in Fig. 2.5.

Tissue Oxygen Monitoring Using EPR Spectroscopy 35

22 G Needle (1) LiPc Crystal (Pellet)

Piston

~2–3 mm (3) Push out

Mouse leg (2) Inject

Female C3H

Fig. 2.4. Schematic drawing of the implantation of LiPc crystals (1). A portion of LiPc crystals (5–10 mg) is suspended with an appropriate volume (10–20␮L) of physiologic saline and kneaded until a slurry paste is obtained. Then, the LiPc slurry was placed in the tip of a 20-gauge needle to make a 2–3 mm long, soft pellet (2). The needle is injected into the region of interest of the animal, and (3) the pellet was pushed out using a smooth piston. The average dry weight of the pellet was 0.8± 0.1 mg (mean ± SD).

y = 5.13x + 43.57 R² = 0.998

y = 4.26x + 31.97 R² = 0.999 0

200 400 600 800

0 40 80 120 160 200 pO₂ (mmHg)

Linewidth (mG) RT

37°C

Fig. 2.5. Calibration curve of the EPR linewidth of LiPc versus pO2 under various gas conditions, including 21 (room air), 15, 10, 5, 1.25, and 0% (Ar) oxygen, each with the constant gas flow rate of 700 mL/min. The EPR measurement was done after at least 30 min equilibration under each gas flow condition at 37^◦C (gray circle) or at room temperature (black circle). The EPR signal was measured using a 700 MHz CW EPR spectrometer. The EPR conditions were as follows: microwave frequency: 700 MHz;

scan rate: 0.25 mT/s; time constant: 0.003 s; field modulation frequency: 13.5 kHz. The microwave power (0.005–0.3 mW) and field modulation width (0.01–0.07 mT) were adjusted to avoid signal saturation and line broadening.

3.4. In Vivo EPR Measurement 3.4.1. The Animal Setting

The animal setting for in vivo CW and TD EPR measurement is basically same.

1. Mice are anesthetized with 1.5% isoflurane in medical air (flow rate 700 mL/min) and placed in a mouse holder (see Note 6).

2. The mouse legs and lower abdomen are restrained by adhe-sive tape to the holder. (see Note 7).

3. Before inserting the temperature probe into the rectum, the tip of non-magnetic temperature probe (FISO; FOT-L-SD, Fiber Optic sensor) is coated with gel.

4. The surface coil is placed on the region where the LiPc crys-tal is implanted.

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5. Animal is positioned to resonator in the EPR magnet.

6. EPR measurement is initiated using the minimum modula-tion amplitude and power. (see Note 8)

7. Data processing is done with the calibration curve.

3.4.2. CW EPR Conditions

Microwave frequency= 700 MHz, scan rate = 0.25 mT/s, time constant = 0.003 s, field modulation frequency: 13.5 kHz. The microwave power= 0.005–0.3 mW and field modulation width (0.01–0.07 mT).

Figure 2.6shows an example of CW EPR data of time course of the pO₂levels in mouse femoral and mammary pad during and after breathing carbogen (95% O₂, 5% CO₂; see Note 9). The pO₂ levels in both the studied tissues increase gradually during carbogen breathing in mice, and decrease when the animals are switched back to breathing room air.

Carbogen air

0 10 20 30 40 50 60 70

–10 0 20 40 60 80 100 120 Time (min)

pO2 (mmHg)

air

Femoral muscle Mammary pad

Fig. 2.6. Time course of pO2in the femoral muscle and mammary pad before, during, and after the mouse breathed carbogen. Values are indicated as mean± SE of average values of 3 mice each, every 10 min. The pO2levels in both sites increased gradually when the mice were exposed to carbogen. When the breathing gas was switched back to room air, the pO₂ levels went down faster in the femoral muscle compared to the mammary pad. The pO2 levels in the mammary tissue are lower than in the femoral muscle.

3.4.3. TD EPR As an oxygen sensor for TD EPR oxymetry, LiPc crystal pos-sesses all the desirable properties including high spin density, sin-gle spectrum, very narrow line width (long transversal relaxation time T2∗), high sensitivity to changes in oxygen concentration, and stability in biological tissues. The line width of EPR spectrum is inversely related to the decay constant of TD EPR signal after irradiation pulse which must be longer than the recovery time of receiver system.

TD EPR conditions: resonant frequency = 750 MHz; exci-tation pulse width= 125 ns; TR = 7.5 ␮s; No. of sampling = 1,760. The dead-time of the spectrometer was estimated as 270 ns.

Tissue Oxygen Monitoring Using EPR Spectroscopy 37

Fig. 2.7. The FT spectra of the oxygen-sensitive LiPc batch, using a time-domain RF EPR spectrometer at two different oxygen percentages, 0% O2and 2.04% O2, respectively.

A 1-GHz sampler with a channel limitation of 4 k points, corresponding to 4.096 ms was used to collect the data. The wiggles in the 0% O2data are due to truncation of the FID at 4.1 ms, no digital filters are used.

Typical TD EPR spectra are shown in Fig. 2.7 under two different pO₂ levels (0% O₂ and 2.4% O₂) for the same batch whose CW spectra and the oxygen responses as shown in Fig. 2.2.

For example, time-course pO₂change after carbogen breathing in normal muscle and tumor are shown in Fig. 2.8. The basal pO₂ values in muscle region are higher than those of tumor region. In addition, the response to carbogen breathing in the tumor region is low.

0 5 10 15 20 25

0 10 20 30 40 50 Time (min)

pO2 (mmHg)

Air Carbogen (nomal muscle)

0 1 2 3 4 5 6 7 8 9

0 10 20 30 40 Time (min)

pO2 (mmHg)

Air Carbogen (Tumor) )

b ( )

a (

Fig. 2.8. Time course of pO2in the femoral muscle and tumor before and after the mouse breathed carbogen.

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