The strong magnetic moment of the unpaired electron promotes both spin lattice and spin-spin relaxation of the surrounding water protons, resulting with decrease in their spin-lattice (T1) and spinspin (T2) relaxation times or enhancement in signal intensity on T1 or T2 weighted MR images. Relaxation constants of (MGD)2-Fe(II)-NO, (MGD)2-Fe(II) at 500 MHz\* and 300 MHz\*\* is shown indicating that relaxivity is not magnetic field dependent over this range.

Table 2. Relaxation constants of biosensor complexes are shown for NO detection in aqueous solutions.

Inhibition of Nitric Oxide Synthase Gene Expression:

hormone molecules in tissue.

reference Fujii, et al. 2007.

MR contrast due to trapped NO can be expressed in Eq 2, 3:

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 129

1/T1[NO-Fe(II)(MGD)2] = 1/T1(observed) – 1/T\* = r1*i*[NO-Fe(II)(MGD)2] × concentration (2)

 1/Tobs = 1/T*\**+ r1*i*[NO-Fe(II)(MGD)2]; *i* = 1, 2... (3) where T1(observed) is long longitudinal relaxation time constant and T1\* is short longitudinal relaxation time constant in absence of [NO-Fe(II)(MGD)2], r1i is longitudinal relaxivity dpends on I = 1, 2, 3 the number of carbons in branch of MGD chain, [NO-Fe(II)(MGD)2], concentration and interaction with NO specific bioactive receptor, enzyme, antibody,

Fig. 5. Imaging of preformed (MGD)2-Fe(II)-NO complex. Transverse T1-weighted MR images in the axial plane of the liver of Wistar rats. Two ml of 9.5 mM (MGD)2-Fe(II)-NO complex was injected i.p. in the lower abdomen of 250 g rats (n = 3). **a:** Control, before injection; **b:** 60 min after injection of (MGD)2-Fe(II)-NO complex. A sketch at bottom shows the interaction of No with heme as basis of MRI signal. Reproduced with permission for

In other study, NO complex was prepared from NO gas and (MGD)2-Fe(II) in glass capillaries. The strong magnetic moment of the unpaired electrons promote both spin lattice and spin-spin relaxation of the surrounding water protons and show a decrease in their spin-lattice (T1) and spin-spin (T2) relaxation constants or show enhanced signal intensity in T1 or T2 weighted MR images as shown in Table 2 and Figure 5 [Hortelano, et al. 2005]. Major points were:

In previous study, (MGD)2-Fe(II)-NO and LPS complexes were used as in vivo MR contrast agents [Fujii, et al. 2007]. First, intraperitoneal injection of 2 ml of 9.1 mM (MGD)2-Fe(II)-NO complex (150 nmol/g tissue) showed relative difference on T1-weighted MR images of rat liver site before and after injection of NO complex as shown in Figure 5.

 The images indicated the NO complex very effective ''intrinsic contrast agent,'' to enhance the contrast in several other organs.

The Second, LPS injected animals after 6 hours of (MGD)2-Fe(II) injection, showed subsequent MRI detection and reproducible visualization of NO generated in liver of animals as shown in Figure 5 and Fig. 6a (indicated by the arrow) as plots against time with the intensity of the reference water sample. Figure 6b showed that the image intensity in the liver increased with the NO generation, without any intensity change in reference sample in the presence of the specific NOS inhibitor *N*-monomethyl L-arginine (L-NMMA).

Interestingly, other face of (MGD)2-Fe(II)-NO complex is 'spin trap'. Spin traps are chemical compounds used to detect unstable free radicals such as hydroxyl (\*OH) and superoxide (O2 \*-) in biological systems. Spin trapping compounds (spin traps) selectively react with NO and trap the nitric oxide. The spin-trap stock (MGD)2-Fe(II) complex solution (mixture of deoxygenated saline MGD (100 mM) and FeSO4 (20 mM) solutions in nitrogen) makes (MGD)2-Fe(II)-NO complex by mixing (MGD)2-Fe(II) with either pure NO gas or *S*-nitroso-*N*acetyl DL-penicillamine (SNAP). The pure NO gas is prepared by passing NO gas through a KOH solution. Major concern was the coordination of NO to the Fe complex altered the solubility of N0-Fe(II)(MGD)2 < 1 mM. The in vivo half life of N0-Fe(II)(MGD)2 was 41 minutes and only 40% trapped NO was estimated by detection of Fe(II)(MGD)2 induced inhibited NOS and decrease in NO formation and perfusion in the tissue [Fichtlscherer, et al. 2000].

$$\begin{aligned} \text{2NO} + \text{Fe(III)(MGD)} & \xrightarrow{\text{H}\_2\text{O} \text{ (\*OH)}} \text{NO-Fe(II)(MGD)} + \text{NO}\_2 \\\\ \text{NO} + \text{Fe(III)(MGD)} & \xrightarrow{\text{Reducing Equivalent}} \text{NO-Fe(II)(MGD)} \text{2(40\%)} \end{aligned} \tag{1}$$

In other study, NO complex was prepared from NO gas and (MGD)2-Fe(II) in glass capillaries. The strong magnetic moment of the unpaired electrons promote both spin lattice and spin-spin relaxation of the surrounding water protons and show a decrease in their spin-lattice (T1) and spin-spin (T2) relaxation constants or show enhanced signal intensity in T1 or T2 weighted MR images as shown in Table 2 and Figure 5 [Hortelano, et al. 2005].

The relaxation constants of (MGD)2-Fe(II)-NO in aqueous media were characteristic and

T1 and T2 relaxation constants of liver were measured 1.17(1/mM \* sec) and 1.32

 T1 relaxation constant of (MGD)2-Fe(II), was reported 0.044 (1/mM \* sec) at both 20 MHz and 85 MHz magnetic fields. The T1 relaxation constant was field independent; Distinct increase in relaxivity after complexing NO with (MGD)2-Fe(II). These unique properties of complex suggested its feasibility to visualize the regional distribution in

In previous study, (MGD)2-Fe(II)-NO and LPS complexes were used as in vivo MR contrast agents [Fujii, et al. 2007]. First, intraperitoneal injection of 2 ml of 9.1 mM (MGD)2-Fe(II)-NO complex (150 nmol/g tissue) showed relative difference on T1-weighted MR images of rat

The images indicated the NO complex very effective ''intrinsic contrast agent,'' to

The Second, LPS injected animals after 6 hours of (MGD)2-Fe(II) injection, showed subsequent MRI detection and reproducible visualization of NO generated in liver of animals as shown in Figure 5 and Fig. 6a (indicated by the arrow) as plots against time with the intensity of the reference water sample. Figure 6b showed that the image intensity in the liver increased with the NO generation, without any intensity change in reference sample in

Interestingly, other face of (MGD)2-Fe(II)-NO complex is 'spin trap'. Spin traps are chemical compounds used to detect unstable free radicals such as hydroxyl (\*OH) and superoxide (O2

in biological systems. Spin trapping compounds (spin traps) selectively react with NO and trap the nitric oxide. The spin-trap stock (MGD)2-Fe(II) complex solution (mixture of deoxygenated saline MGD (100 mM) and FeSO4 (20 mM) solutions in nitrogen) makes (MGD)2-Fe(II)-NO complex by mixing (MGD)2-Fe(II) with either pure NO gas or *S*-nitroso-*N*acetyl DL-penicillamine (SNAP). The pure NO gas is prepared by passing NO gas through a KOH solution. Major concern was the coordination of NO to the Fe complex altered the solubility of N0-Fe(II)(MGD)2 < 1 mM. The in vivo half life of N0-Fe(II)(MGD)2 was 41 minutes and only 40% trapped NO was estimated by detection of Fe(II)(MGD)2 induced inhibited NOS

\*-)

(1)

the presence of the specific NOS inhibitor *N*-monomethyl L-arginine (L-NMMA).

and decrease in NO formation and perfusion in the tissue [Fichtlscherer, et al. 2000].

2NO + **Fe(III)**(MGD)2 H2O (\*OH)\_\_\_\_\_\_\_ NO-**Fe(II)**(MGD)2 + NO2-

NO + **Fe(III)**(MGD)2 \_\_\_\_Reducing Equivalent\_\_\_\_ NO-**Fe(II)(**MGD)2 (40%)

(1/mM \* sec) respectively at 500 MHz at increasing concentration of NO;

liver site before and after injection of NO complex as shown in Figure 5.

Major points were:

complex concentration dependent;

tissue *in vivo* where the NO was trapped.

enhance the contrast in several other organs.

MR contrast due to trapped NO can be expressed in Eq 2, 3:

$$\text{1/} \text{Tl} \text{[NO-Fe(II)(MGD)} \text{]} = \text{1/} \text{Tl} \text{(cherved)} - \text{1/} \text{T\*} = \text{r1} \text{[NO-Fe(II)(MGD)} \text{]} \times \text{concentration} \quad \text{(2)}$$

$$1/\Gamma\_{\rm obs} = 1/\Gamma^\* + \text{r1}\_i [\text{NO-Fe(II)(MGD)}\_2]; i = 1, 2... \tag{3}$$

where T1(observed) is long longitudinal relaxation time constant and T1\* is short longitudinal relaxation time constant in absence of [NO-Fe(II)(MGD)2], r1i is longitudinal relaxivity dpends on I = 1, 2, 3 the number of carbons in branch of MGD chain, [NO-Fe(II)(MGD)2], concentration and interaction with NO specific bioactive receptor, enzyme, antibody, hormone molecules in tissue.

Fig. 5. Imaging of preformed (MGD)2-Fe(II)-NO complex. Transverse T1-weighted MR images in the axial plane of the liver of Wistar rats. Two ml of 9.5 mM (MGD)2-Fe(II)-NO complex was injected i.p. in the lower abdomen of 250 g rats (n = 3). **a:** Control, before injection; **b:** 60 min after injection of (MGD)2-Fe(II)-NO complex. A sketch at bottom shows the interaction of No with heme as basis of MRI signal. Reproduced with permission for reference Fujii, et al. 2007.

Inhibition of Nitric Oxide Synthase Gene Expression:

(PARAVISION 3.2).

detecting solid tissues.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 131

experiments, anesthesia was administered as sodium pentobarbital (30 mg/kg, i.p.) and ketamine (20 mg/kg, intra-muscular injection) before injection of contrast agent in animals. For imaging, animals were anesthetized by intubating with 14 gauge, 2 inch intravenous catheter (Abbocath Lab, IL) on nose with 30% oxygen/70% nitrogen mixture containing 5% isoflurane/air mixture to continuous supply through nose during MRI session. Animals were kept in vertical direction to the side of MRI gantry and 30 mm diameter RF insert covering kidneys in the center of magnet. During six hours after administration of LPS to Wistar rats (150–200 g) they were injected intraparitoneally with 3 ml of (MGD)2-Fe(II) [MGD:100 mM, Fe:20 mM] at different intervals. MR images of the anesthetized rats (n = 5) were obtained after 1 hr spin trap injection with a Bruker Biospin 500 MHz scanner

Different imaging techniques are available in our lab on 500 MHz and 900 MHz scanners. In vivo NO induction was carried out according to previously reported methods with slight

 Axial T2-weighted spin echo sequence with fat suppression (TR 2000 ms, TE 100 ms, flip angle 30; EC 1/1 15.6 kHz) was used for more detailed T2-weighted information in

 Axial T1-weighted images were acquired at TR/TE/flip angle = 750 ms/ 4.18 ms/25º, FOV/matrix size/spatial resolution = 2.6 x 1.4 cm2/ 256 x 256 ×75 μm, and the inversion time (TI approximately 250 ms) set to null the normal the background tissue. Gradient echo sequence in-phase and opposed-phase(TR 180 ms, TE 2.3 ms/4.5 ms, flip angle 30 were done as a dual echo sequence to show renal lesions hypointense and parenchyma isointense with hyperintense protein rich cyst. Opposed phased T1-

 Axial T1-weighted gradient echo sequence for dynamic perfusion/ diffusion weighted imaging (TR 130 ms, TE 1.0 ms, flip angle 30) was done after 30 ml intravenous gadolinium contrast injection for acquiring pre-contrast and post-contrast images in arterial phase to distinguish perfusion in the tissues. Parameters for T1-weighted spinecho images were TR 1500 msec, TE 10 msec, 2 NEX, 1-mm slice thickness, 0.5-mm slice

The original bird-cage coil with 2.6 cm diameter Rf insert was used for MR imaging. We established in lab MR image analysis by shareware software NIH Image J, a PC version supplied by Scion Corporation. The analysis of averaged image intensities from selected regions of interest 15 x 3 x 15, and matrix , 256 x 256 served as best for measurement of signal enhancement. An external reference (saline solution in a sealed test tube) placed near the animal serves to normalize intensities from both control and test groups. Percentage enhancements can be calculated from the normalized data [35]. Functional dependence of the MR imaging signal intensity *S* on the intrinsic properties (proton spin density *N*(*H*), T1, T2) has been derived based on the assumption of a monoexponential dependence on T1 and T2 with a spin-echo sequence: *S*(TE,TR) = *N*(*H*)exp(-TE/T2) [1 -exp[-(TR/ TE)/T1]]. The term on the right reflects the T1 dependence, and the exponential expression on the left reflects the T2 effects. TE << T2 generates a T1-weighted. T1 image is obtained by means of least-squares fitting (Marquardt-Levenberg algorithm) of the mean intensity values versus

modifications [Lai, et al. 1994, Yoshimura, et al. 1996, Green, et al. 1998].

weighted gradient echo sequences were sensitive to fat.

gap, field of view 12 x 3 x 12 cm3, and matrix, 256 x 256.

Fig. 6. Imaging of NO in six phantoms and LPS-treated rats. **a:** Transverse T1-weighted MR images focussed on a selected region of the liver in LPS-doped rats. The MR images were measured at the times indicated. Six hours after LPS injection, the NO spin-trap [3 ml of (MGD)2-Fe(II), MGD: 100 mM, Fe: 20 mM] was administered i.p. **b:** Plot of MR image intensity with time. Signal intensities were averaged over the selected region indicated by the arrow in a (7\*7 mm2) for three different animals (filled symbols), normalized to intensity of a reference (water in a tube placed next to the animal, open symbols). At zero time, the spin-trap, (MGD)2- Fe(II), was added. **c:** Comparison of the signal intensity in a selected slice region of the liver in the presence and absence of the NOS inhibitor, L-NMMA. The image intensity in the liver was first normalized to the external standard noted above. The averaged intensity in the selected regions (7 x 7 mm2) was averaged (n = 3). L-NMMA (50 mg/kg in saline) was injected i.p. 3 h after LPS injection (Reproduced from reference Fujii et al. 2007).

#### **2.4 MRI measurement protocol design**

We developed a MRI protocol to investigate the regional distribution of (MGD)2-Fe(II)-NO in various organs. For whole animal imaging, protocols for relaxivity of (MGD)2-Fe(II)-NO were designed on a Bruker 500 MHz, to achieve millimolar relaxivity. For the animal

Fig. 6. Imaging of NO in six phantoms and LPS-treated rats. **a:** Transverse T1-weighted MR images focussed on a selected region of the liver in LPS-doped rats. The MR images were measured at the times indicated. Six hours after LPS injection, the NO spin-trap [3 ml of (MGD)2-Fe(II), MGD: 100 mM, Fe: 20 mM] was administered i.p. **b:** Plot of MR image intensity with time. Signal intensities were averaged over the selected region indicated by the arrow in a (7\*7 mm2) for three different animals (filled symbols), normalized to intensity of a reference (water in a tube placed next to the animal, open symbols). At zero time, the spin-trap, (MGD)2- Fe(II), was added. **c:** Comparison of the signal intensity in a selected slice region of the liver in the presence and absence of the NOS inhibitor, L-NMMA. The image intensity in the liver was first normalized to the external standard noted above. The averaged intensity in the selected regions (7 x 7 mm2) was averaged (n = 3). L-NMMA (50 mg/kg in saline) was injected i.p. 3 h

We developed a MRI protocol to investigate the regional distribution of (MGD)2-Fe(II)-NO in various organs. For whole animal imaging, protocols for relaxivity of (MGD)2-Fe(II)-NO were designed on a Bruker 500 MHz, to achieve millimolar relaxivity. For the animal

after LPS injection (Reproduced from reference Fujii et al. 2007).

**2.4 MRI measurement protocol design**

experiments, anesthesia was administered as sodium pentobarbital (30 mg/kg, i.p.) and ketamine (20 mg/kg, intra-muscular injection) before injection of contrast agent in animals. For imaging, animals were anesthetized by intubating with 14 gauge, 2 inch intravenous catheter (Abbocath Lab, IL) on nose with 30% oxygen/70% nitrogen mixture containing 5% isoflurane/air mixture to continuous supply through nose during MRI session. Animals were kept in vertical direction to the side of MRI gantry and 30 mm diameter RF insert covering kidneys in the center of magnet. During six hours after administration of LPS to Wistar rats (150–200 g) they were injected intraparitoneally with 3 ml of (MGD)2-Fe(II) [MGD:100 mM, Fe:20 mM] at different intervals. MR images of the anesthetized rats (n = 5) were obtained after 1 hr spin trap injection with a Bruker Biospin 500 MHz scanner (PARAVISION 3.2).

Different imaging techniques are available in our lab on 500 MHz and 900 MHz scanners. In vivo NO induction was carried out according to previously reported methods with slight modifications [Lai, et al. 1994, Yoshimura, et al. 1996, Green, et al. 1998].

The original bird-cage coil with 2.6 cm diameter Rf insert was used for MR imaging. We established in lab MR image analysis by shareware software NIH Image J, a PC version supplied by Scion Corporation. The analysis of averaged image intensities from selected regions of interest 15 x 3 x 15, and matrix , 256 x 256 served as best for measurement of signal enhancement. An external reference (saline solution in a sealed test tube) placed near the animal serves to normalize intensities from both control and test groups. Percentage enhancements can be calculated from the normalized data [35]. Functional dependence of the MR imaging signal intensity *S* on the intrinsic properties (proton spin density *N*(*H*), T1, T2) has been derived based on the assumption of a monoexponential dependence on T1 and T2 with a spin-echo sequence: *S*(TE,TR) = *N*(*H*)exp(-TE/T2) [1 -exp[-(TR/ TE)/T1]]. The term on the right reflects the T1 dependence, and the exponential expression on the left reflects the T2 effects. TE << T2 generates a T1-weighted. T1 image is obtained by means of least-squares fitting (Marquardt-Levenberg algorithm) of the mean intensity values versus

Inhibition of Nitric Oxide Synthase Gene Expression:

functional MRI [Caramia, et al. 1998].

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 133

Fig. 7. NO sensitive biomarker imaging contrast agents. Example of NO-Fe(II)(DTC)2 is cited to illustrate the signal change during changes in cerebral blood flow and possibility of

Two decades ago, NO activation-induced signal changes in functional MRI (fMRI) was observed [Di Salle, et al. 1997]. Hb–NO was found to produce marked stimulator dose and concentration-dependent NO sensitive fMRI signal intensity changes (increased after aqueous NO solution, nitrite, or dithionite and nitrite added to blood while it decreased on the addition of ascorbic acid) examined by T1-, T2-, and T2 -weighted MRI in venous erythrocytes with possibility of physiological MRI evaluation associated with NO change [Di Salle, et al. 1997]. Transient formation of two paramagnetic species (met Hb and NO-Hb) enhanced the signal intensity, while ascorbate reduced the signal intensity in fMRI experiment on healthy volunteers during standard tasks. Further, infusion of NO precursor, L-arginine increased the cerebral blood volume (measured by MRI) in nonischemic spontaneously hypertensive rats [Foster, et al. 1998]. In other study, administration of an NO donor, isosorbide dinitrate (ISDN) increased both tumor blood flow and partial oxygen pressure in mice implanted with liver tumor in the thigh [Jordan, et al. 2000]. These findings indicate blood flow-independent change in MRI signal produced by different additives or stimulators. These observations open a new perspective on the monitoring of NO and the in vivo speculation of NO effects to test new drugs by using magnetic resonance techniques. Fuji et al. 1999 first time reported *in vivo* MRI of NO using Fe(MGD)2 as NO–Fe(MGD)2 spintrapping technique in a septic shock rat model [Fujii, et al. 1999]. (Fig. 6A). The reduced signal after administration of an iNOS inhibitor confirmed the MRI visibility of NO–

**NO-Fe(II)(DTC)2**

TR. T2 weighted image is obtained by TR >> T2. Mean intensity values are fitted versus the TE (Marquardt Levenberg algorithm).

T1 weitghted MR contrast due to trapped NO as NO-Fe(II)X spin trap in tissue can be expressed in Eq 4, 5:

$$1/\text{T1}[\text{NO-Fe(II)X}] = 1/\text{T1}\_{\text{(observed)}} - 1/\text{T} = \text{r1}\_{i}[\text{NO-Fe(II)X}] \times \text{concentration} \tag{4}$$

$$1/\Gamma\_{\rm obs} = 1/\Gamma^\* + \text{r1}\_i [\text{NO-Fe(II)}\lambda]; i = 1, 2... \tag{5}$$

where T1(observed) is long longitudinal relaxation time constant and T1\* is short longitudinal relaxation time constant in absence of [NO-Fe(II)X],

r1i is longitudinal relaxivity dpends on i= 1, 2, 3 the number of carbons in branch of X contrast agent, its concentration and interaction with NO specific bioactive receptor, enzyme, antibody, hormone molecules in tissue.

#### **2.5 Approaches to NO evaluation by Magnetic Resonance Imaging (MRI) techniques**

Two major developments reported in context with NO. First, a search of NO specific stable multimodal MR/EPR sensitive spin trap complex; second, calibrating imaging characteristics of spin trap complex on NMR/MRI/PEDRI techniques. NO is perfused in gaseous phase or remains in ionic phase by redox state in the tissues. Both forms of NO are captured or trapped by Fe(II)-(DTC)2 complexes and show MRI signal change due to reactive nitrosylation (reaction of intracellular NO with Fe(II)(DTC)2 to form NO-Fe(II)(DTC)2 complex) shown in Eq 6. Second-order rate constant of the reaction of NO with Fe(II)(ProDTC)2 was reported to be (1.1 0.3)×108 M-1 s-1[Caramia, et al. 1998].

$$\text{Fe(II)(DTC)}\_2 \blackheadrightarrow \text{Fe(II)(ProDTC)}\_2 + \text{NO} \twoheadrightarrow \text{NO-Fe(II)(DTC)}\_2 \tag{6}$$

Recently, attempts have been made to overcome the limitations of EPR-based imaging with low resolution and large sample size by employing new approaches of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) techniques. Several paramagnetic metal complexes have been widely used as contrast agents in magnetic resonance imaging (MRI) due to their ability to enhance the NMR relaxation of neighboring protons [von Bohlen, et al. 2003]. Paramagnetic NO-Fe-DTC complex has been utilized to visualize NO generated in living rats with septic shock [Fichtlscherer, et al. 2000, Fujii, et al. 1999]. Paramagnetic nitrosyl iron complex showed contrast properties to enhance the signal intensity of SNP-perfused rat liver in MRI [Fichtlscherer, et al. 2000, Fujii, et al. 1999]. Thus, a paramagnetic nitrosyl iron complex may be potentially useful as a microscopic localizer MRI contrast agent specific for NO accumulation in living organisms. Other protonelectron-double-resonance-imaging (PEDRI) technique was based on the enhancement of proton NMR signal intensity in the presence of radicals through the Overhauser effect Mulsch et al. 1999,Foster, et al.1998]. Rat livers were exposed to SNP as an NO donor exhibited intense PEDRI images [Rast, et al. 2001, Jordan, et al.2004]. PEDRI imaging is a potential tool to study NO in large biological specimens. Still imaging application of MR and EPR spectroscopy using nitric oxide synthase inhibitors, oxygenation or perfusion are in infancy [von Bohlen, et al. 2003, Zhou, et al. 2009, Nagano, et al. 2002, Anbar, 2000]

TR. T2 weighted image is obtained by TR >> T2. Mean intensity values are fitted versus the

T1 weitghted MR contrast due to trapped NO as NO-Fe(II)X spin trap in tissue can be

1/T1[NO-Fe(II)X] = 1/T1(observed) – 1/T\* = r1*i*[NO-Fe(II)X] × concentration (4)

 1/Tobs = 1/T*\**+ r1*i*[NO-Fe(II)X]; *i* = 1, 2... (5) where T1(observed) is long longitudinal relaxation time constant and T1\* is short longitudinal

r1i is longitudinal relaxivity dpends on i= 1, 2, 3 the number of carbons in branch of X contrast agent, its concentration and interaction with NO specific bioactive receptor,

**2.5 Approaches to NO evaluation by Magnetic Resonance Imaging (MRI) techniques**  Two major developments reported in context with NO. First, a search of NO specific stable multimodal MR/EPR sensitive spin trap complex; second, calibrating imaging characteristics of spin trap complex on NMR/MRI/PEDRI techniques. NO is perfused in gaseous phase or remains in ionic phase by redox state in the tissues. Both forms of NO are captured or trapped by Fe(II)-(DTC)2 complexes and show MRI signal change due to reactive nitrosylation (reaction of intracellular NO with Fe(II)(DTC)2 to form NO-Fe(II)(DTC)2 complex) shown in Eq 6. Second-order rate constant of the reaction of NO with

 Fe(II)(DTC)2Fe(II)(ProDTC)2 + NO NO-Fe(II)(DTC)2 (6) Recently, attempts have been made to overcome the limitations of EPR-based imaging with low resolution and large sample size by employing new approaches of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) techniques. Several paramagnetic metal complexes have been widely used as contrast agents in magnetic resonance imaging (MRI) due to their ability to enhance the NMR relaxation of neighboring protons [von Bohlen, et al. 2003]. Paramagnetic NO-Fe-DTC complex has been utilized to visualize NO generated in living rats with septic shock [Fichtlscherer, et al. 2000, Fujii, et al. 1999]. Paramagnetic nitrosyl iron complex showed contrast properties to enhance the signal intensity of SNP-perfused rat liver in MRI [Fichtlscherer, et al. 2000, Fujii, et al. 1999]. Thus, a paramagnetic nitrosyl iron complex may be potentially useful as a microscopic localizer MRI contrast agent specific for NO accumulation in living organisms. Other protonelectron-double-resonance-imaging (PEDRI) technique was based on the enhancement of proton NMR signal intensity in the presence of radicals through the Overhauser effect Mulsch et al. 1999,Foster, et al.1998]. Rat livers were exposed to SNP as an NO donor exhibited intense PEDRI images [Rast, et al. 2001, Jordan, et al.2004]. PEDRI imaging is a potential tool to study NO in large biological specimens. Still imaging application of MR and EPR spectroscopy using nitric oxide synthase inhibitors, oxygenation or perfusion are in

Fe(II)(ProDTC)2 was reported to be (1.1 0.3)×108 M-1 s-1[Caramia, et al. 1998].

infancy [von Bohlen, et al. 2003, Zhou, et al. 2009, Nagano, et al. 2002, Anbar, 2000]

TE (Marquardt Levenberg algorithm).

relaxation time constant in absence of [NO-Fe(II)X],

enzyme, antibody, hormone molecules in tissue.

expressed in Eq 4, 5:

#### **NO-Fe(II)(DTC)2**

Fig. 7. NO sensitive biomarker imaging contrast agents. Example of NO-Fe(II)(DTC)2 is cited to illustrate the signal change during changes in cerebral blood flow and possibility of functional MRI [Caramia, et al. 1998].

Two decades ago, NO activation-induced signal changes in functional MRI (fMRI) was observed [Di Salle, et al. 1997]. Hb–NO was found to produce marked stimulator dose and concentration-dependent NO sensitive fMRI signal intensity changes (increased after aqueous NO solution, nitrite, or dithionite and nitrite added to blood while it decreased on the addition of ascorbic acid) examined by T1-, T2-, and T2 -weighted MRI in venous erythrocytes with possibility of physiological MRI evaluation associated with NO change [Di Salle, et al. 1997]. Transient formation of two paramagnetic species (met Hb and NO-Hb) enhanced the signal intensity, while ascorbate reduced the signal intensity in fMRI experiment on healthy volunteers during standard tasks. Further, infusion of NO precursor, L-arginine increased the cerebral blood volume (measured by MRI) in nonischemic spontaneously hypertensive rats [Foster, et al. 1998]. In other study, administration of an NO donor, isosorbide dinitrate (ISDN) increased both tumor blood flow and partial oxygen pressure in mice implanted with liver tumor in the thigh [Jordan, et al. 2000]. These findings indicate blood flow-independent change in MRI signal produced by different additives or stimulators. These observations open a new perspective on the monitoring of NO and the in vivo speculation of NO effects to test new drugs by using magnetic resonance techniques. Fuji et al. 1999 first time reported *in vivo* MRI of NO using Fe(MGD)2 as NO–Fe(MGD)2 spintrapping technique in a septic shock rat model [Fujii, et al. 1999]. (Fig. 6A). The reduced signal after administration of an iNOS inhibitor confirmed the MRI visibility of NO–

Inhibition of Nitric Oxide Synthase Gene Expression:

MRI technique [Fujii, et al.1999].

**3.2 Emission enhancement mechanism**

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 135

contrast in the vascular structure, such as the hepatic vein and inferior vena cava, which can be reduced after administration of an iNOS inhibitor.Initially, NO mapping was reported by whole body EPR imaging at poor resolution [Claudette, et al. 2005,Yoshimura, et al. 1996]. Recently, MRI strategies combined with in vivo spin-trapping map ''NO distributions'' within tissues and organs showed much higher spatial resolution using (MGD)2-Fe(II)-NO as a NMR contrast agent in vivo [Fichtlscherer, et al. 2000]. The MR images also provided follow-up of NO generation kinetics at different sites within the organ at much higher spatial resolution than with EPR [Claudette, et al. 2005,Yoshimura, et al. 1996]. NO makes stable complex such as (MGD)2-Fe(II)-NO. In vivo, hemoglobin is normally the natural NO spin-trap such that NO tends to bind with hemoglobin or oxidize the hemoglobin, followed by conversion to nitrosyl-hemoglobin or methemoglobin, both of these complexes are paramagnetic. Authors believe that during brain stimulation, NO is generated in some regions and it is quite possible that (paramagnetic) nitrosyl-hemoglobin and methemoglobin are formed in these active regions in brain. The MRI signal intensity enhancement in these regions results from changes in blood flow rich with these complexes. However, for blood flow–independent effects in MRI, the paramagnetic relaxation from spin-trapped NO might provide a new fMRI contribution in future. To our knowledge, there have been no reports successfully imaging or detecting free radicals such as NO in vivo by NMR or fMRI. Further, both short lifetime and rapid diffusion of NO in time preclude any effective relaxation enhancement mechanisms. NMR spin trapping serves to overcome these problems such as NO short lifetime and its fast diffusion in tissues to some extent. In other classic study on rats, spin relaxation constants of dinitrosyl-iron bound albumin or GSH and mononitrosyl-iron dithiocarbamate MGD complexes showed concentration dependence by

Present state of art in NO detection was developed over last 2 decades using fluorescein cheletropic compounds visualized by emission enhancement and photoinduced electron transfer mechanisms. Fluorescence properties of fluorescein derivatives are controlled by the photoinduced electron transfer (PET) process from the benzoic acid moiety to the fluorophore, the xanthene ring. PET is a widely accepted mechanism for fluorescence quenching in which electron transfer from the PET donor to the excited fluorophore diminishes the fluorescence of the fluorophore. A brief survey of these compounds and their fluorescence mechanisms will catch the attention of readers with the newer developments of NO detection in tissues. EPR (also known as electron spin resonance or ESR) explores the magnetic moment associated with the unpaired electron(s) in free radicals and paramagnetic metal ions [Mader, 1998, Swartz, 2007]. It has been applied for the detection of free radicals and paramagnetic metal ions in various biological conditions [Berliner, et al. 2004]. When an unpaired electron in a paramagnetic molecule is subjected to an external magnetic field, the energy level of the electron splits into two quantum states with different energy levels. Typically, the magnetic field is scanned with a fixed microwave frequency of approximately 9.5 GHz (usually called the X-band). Specimens less than about 100 μL are measurable in an Xband spectrometer. However, larger biological samples (e.g., tissues, organs, and live animals) cannot be measured with a conventional X-band spectrometer due to the high dielectric loss of water at such frequencies and the small size of the EPR cavity resonator.

Fe(MGD)2 complex especially in inflammation which has high levels of NO. Now many MRI visible nitrosyl-iron complexes are known for MRI mapping of NO using a conventional 1.5 T MR scanner. The principle is that after exposure to NO in tissues Fe(MGD)2 imaging agents form nitrosyl-iron complexes, which shorten the T1 and T2 relaxation time in a concentration-dependent manner [Hong, et al. 2009]. Mainly, four major techniques are reported in quest of developing new NO sensitive MRI contrast agents. First, unpaired electrons of the nitrosyl–iron complexes can enter into dynamic nuclear polarization with water protons, a technique called proton-electron-double-resonance imaging (PEDRI). Second, superparamagnetic iron oxide (SPIO) or ferumoxides dephase T2 weighted MRI signal dependent on SPIO: NO donor ratios indicating inverse relation of contrast enhancement by SPIO with increasing levels of NO in tissue. The MRI signal change was due to reduction of ferric to ferrous iron with result of decrease in paramagnetic relaxation of water protons [Kojima, et al.2001]. Third, technical development emerged as irreversible paramagnetic chemical exchange saturation transfer (PARACEST) based MRI contrast agents in imaging [Liu, et al. 2007, Zhang, et al. 2003, Wu, et al. 2008]. A PARACEST MRI agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) orthoaminoanilide (Yb-DO3A-OAA), was developed for NO detection (Fig. 6B) [9]. Fourth, NO and O2 combine by irreversible covalent bond that causes a quick disappearance of the PARACEST effect captured in the MR images. PARACEST MRI is fast data acquisition (in few minutes) vs SPIO based imaging is slow (in few hours). PARACEST effect has poor sensitivity (only detects millimolar concentrations of NO).

#### **2.6 Approach of real-time NO synthase inhibition as blood oxygenation dependent MRI signal**

Nitric oxide synthase inhibition (NOSi) in humans by blood oxygenation level-dependent (BOLD) MRI as reduced response to NOSi was reported using BOLD MRI to compare changes in R2\* to direct measures of renal medullary oxygen levels and blood flow using Nnitro-L-arginine methyl ester invasive probes (OxyLite/OxyFlo) to examine for the first time the effect of NOSi on intrarenal oxygenation in humans [Li, et al. 2009, Di Salle, et al. 1997]. Authors showed that NOSi decreased medullary pO2 and blood flow in a dose-dependent manner, and BOLD MRI showed an increase in medullary R2\* consistent with the invasive pO2 measurements.

Other alternate approach of positron emission tomography (PET) was proposed in recent study using nitric oxide imaging probe prepared from [18F] 6-(2-fluoropropyl)-4-methylpyridin-2-amine by substitution at position-6 of substituted 2-amino-4-methylpyridine with favorable properties as a PET tracer to image iNOS activation/expression with PET of animal lungs [Zhou, et al. 2009].

#### **3. Major developments in NO bioimaging probes**

#### **3.1 Magnetic resonance relaxation enhancement mechanism**

In 1999, the first in vivo MRI of NO was reported using the spintrapping technique in a septic shock rat model [Fujii, et al. 1999]. NO was trapped by Fe(MGD)2 in vivo, which can be visualized by MRI. The NO–Fe(MGD)2 complex displayed significantly enhanced

Fe(MGD)2 complex especially in inflammation which has high levels of NO. Now many MRI visible nitrosyl-iron complexes are known for MRI mapping of NO using a conventional 1.5 T MR scanner. The principle is that after exposure to NO in tissues Fe(MGD)2 imaging agents form nitrosyl-iron complexes, which shorten the T1 and T2 relaxation time in a concentration-dependent manner [Hong, et al. 2009]. Mainly, four major techniques are reported in quest of developing new NO sensitive MRI contrast agents. First, unpaired electrons of the nitrosyl–iron complexes can enter into dynamic nuclear polarization with water protons, a technique called proton-electron-double-resonance imaging (PEDRI). Second, superparamagnetic iron oxide (SPIO) or ferumoxides dephase T2 weighted MRI signal dependent on SPIO: NO donor ratios indicating inverse relation of contrast enhancement by SPIO with increasing levels of NO in tissue. The MRI signal change was due to reduction of ferric to ferrous iron with result of decrease in paramagnetic relaxation of water protons [Kojima, et al.2001]. Third, technical development emerged as irreversible paramagnetic chemical exchange saturation transfer (PARACEST) based MRI contrast agents in imaging [Liu, et al. 2007, Zhang, et al. 2003, Wu, et al. 2008]. A PARACEST MRI agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) orthoaminoanilide (Yb-DO3A-OAA), was developed for NO detection (Fig. 6B) [9]. Fourth, NO and O2 combine by irreversible covalent bond that causes a quick disappearance of the PARACEST effect captured in the MR images. PARACEST MRI is fast data acquisition (in few minutes) vs SPIO based imaging is slow (in few hours). PARACEST effect has poor

**2.6 Approach of real-time NO synthase inhibition as blood oxygenation dependent**

Nitric oxide synthase inhibition (NOSi) in humans by blood oxygenation level-dependent (BOLD) MRI as reduced response to NOSi was reported using BOLD MRI to compare changes in R2\* to direct measures of renal medullary oxygen levels and blood flow using Nnitro-L-arginine methyl ester invasive probes (OxyLite/OxyFlo) to examine for the first time the effect of NOSi on intrarenal oxygenation in humans [Li, et al. 2009, Di Salle, et al. 1997]. Authors showed that NOSi decreased medullary pO2 and blood flow in a dose-dependent manner, and BOLD MRI showed an increase in medullary R2\* consistent with the invasive

Other alternate approach of positron emission tomography (PET) was proposed in recent study using nitric oxide imaging probe prepared from [18F] 6-(2-fluoropropyl)-4-methylpyridin-2-amine by substitution at position-6 of substituted 2-amino-4-methylpyridine with favorable properties as a PET tracer to image iNOS activation/expression with PET of

In 1999, the first in vivo MRI of NO was reported using the spintrapping technique in a septic shock rat model [Fujii, et al. 1999]. NO was trapped by Fe(MGD)2 in vivo, which can be visualized by MRI. The NO–Fe(MGD)2 complex displayed significantly enhanced

sensitivity (only detects millimolar concentrations of NO).

**3. Major developments in NO bioimaging probes**

**3.1 Magnetic resonance relaxation enhancement mechanism**

**MRI signal**

pO2 measurements.

animal lungs [Zhou, et al. 2009].

contrast in the vascular structure, such as the hepatic vein and inferior vena cava, which can be reduced after administration of an iNOS inhibitor.Initially, NO mapping was reported by whole body EPR imaging at poor resolution [Claudette, et al. 2005,Yoshimura, et al. 1996]. Recently, MRI strategies combined with in vivo spin-trapping map ''NO distributions'' within tissues and organs showed much higher spatial resolution using (MGD)2-Fe(II)-NO as a NMR contrast agent in vivo [Fichtlscherer, et al. 2000]. The MR images also provided follow-up of NO generation kinetics at different sites within the organ at much higher spatial resolution than with EPR [Claudette, et al. 2005,Yoshimura, et al. 1996]. NO makes stable complex such as (MGD)2-Fe(II)-NO. In vivo, hemoglobin is normally the natural NO spin-trap such that NO tends to bind with hemoglobin or oxidize the hemoglobin, followed by conversion to nitrosyl-hemoglobin or methemoglobin, both of these complexes are paramagnetic. Authors believe that during brain stimulation, NO is generated in some regions and it is quite possible that (paramagnetic) nitrosyl-hemoglobin and methemoglobin are formed in these active regions in brain. The MRI signal intensity enhancement in these regions results from changes in blood flow rich with these complexes. However, for blood flow–independent effects in MRI, the paramagnetic relaxation from spin-trapped NO might provide a new fMRI contribution in future. To our knowledge, there have been no reports successfully imaging or detecting free radicals such as NO in vivo by NMR or fMRI. Further, both short lifetime and rapid diffusion of NO in time preclude any effective relaxation enhancement mechanisms. NMR spin trapping serves to overcome these problems such as NO short lifetime and its fast diffusion in tissues to some extent. In other classic study on rats, spin relaxation constants of dinitrosyl-iron bound albumin or GSH and mononitrosyl-iron dithiocarbamate MGD complexes showed concentration dependence by MRI technique [Fujii, et al.1999].

#### **3.2 Emission enhancement mechanism**

Present state of art in NO detection was developed over last 2 decades using fluorescein cheletropic compounds visualized by emission enhancement and photoinduced electron transfer mechanisms. Fluorescence properties of fluorescein derivatives are controlled by the photoinduced electron transfer (PET) process from the benzoic acid moiety to the fluorophore, the xanthene ring. PET is a widely accepted mechanism for fluorescence quenching in which electron transfer from the PET donor to the excited fluorophore diminishes the fluorescence of the fluorophore. A brief survey of these compounds and their fluorescence mechanisms will catch the attention of readers with the newer developments of NO detection in tissues. EPR (also known as electron spin resonance or ESR) explores the magnetic moment associated with the unpaired electron(s) in free radicals and paramagnetic metal ions [Mader, 1998, Swartz, 2007]. It has been applied for the detection of free radicals and paramagnetic metal ions in various biological conditions [Berliner, et al. 2004]. When an unpaired electron in a paramagnetic molecule is subjected to an external magnetic field, the energy level of the electron splits into two quantum states with different energy levels. Typically, the magnetic field is scanned with a fixed microwave frequency of approximately 9.5 GHz (usually called the X-band). Specimens less than about 100 μL are measurable in an Xband spectrometer. However, larger biological samples (e.g., tissues, organs, and live animals) cannot be measured with a conventional X-band spectrometer due to the high dielectric loss of water at such frequencies and the small size of the EPR cavity resonator.

Inhibition of Nitric Oxide Synthase Gene Expression:

fluorescent probes in NO bioimaging.

Fig. 8. Representative spin-trapping agents for EPR imaging of NO.

Fe(MGD)2 or Fe(DETC)2 have also been tested for EPR imaging to map the spatial distribution of NO generation in the ischemic myocardium [Kuppusamy, et al. 1996]. Subsequently, Fe(MGD)2 was used in both EPR and MR imaging in rats with sepsis shock [Fujii, et al.1999]. It was reported that Fe(MGD)2 can reduce nitrite to NO at physiological pH, which may cause some detection Inaccuracy as shown in Figure 8, although the rate

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 137

AX is almost nonfluorescent [Nagano, et al. 2002]. Thus, a small change in the size of conjugated aromatics, namely, from naphthalene to anthracene, causes a great alteration of the fluorescence properties. Authors compared the HOMO levels of the benzoic acid moieties with xanthine ring and naphthoic acid. Napthoic acid moiety is present in highly fluorescent fluorescein and NX. The HOMO levels of benzoic acid moiety were lower than that of the xanthene ring, while the HOMO level of anthracenecarboxylic acid moiety in fluorescent AX, was higher than that of the xanthene. These results were consistent with the idea that a PET process controls the fluorescence properties of fluorescein derivatives and that these properties can be predicted from the HOMO level of the benzoic acid moiety, with a threshold at around -8.9 eV. This in turn provides a basis for developing novel fluorescence probes with fluorescein-derived structure. Still much remains to justify the

EPR spectroscopy at lower frequencies, the L-band (0.4–1.6 GHz) or S-band(1.6–4 GHz), can be used for in vivo EPR imaging. EPR imaging is generally considered to be the most effective technique for noninvasive observation of the spatial distribution of free radicals [Kleschyov, et al. 2007]. Direct EPR detection of endogenous free radicals in biological samples requires that the radical of interest is present at a concentration higher than the practical detection limit (0.1–0.01 μM) and has a relatively long lifetime. Although such requirement can be met at cryogenic temperature, in vivo detection is almost impossible since most radicals do not have a sufficiently long lifetime. Therefore, spin-trapping techniques have been developed in which the spin traps can react with unstable free radicals to form a relatively stable adduct [Berliner, et al. 2004, Davie, et al. 2004]. The formation of the long lived adduct results in accumulation of steady-state free radicals which can be detected readily by EPR spectroscopy.

In principle, the fluorescence properties of fluorescein derivatives are controlled by the photoinduced electron transfer (PET) process from the benzoic acid moiety to the fluorophore, the xanthene ring. PET is a widely accepted alternate mechanism for fluorescence quenching in which electron transfer from the PET donor to the excited fluorophore diminishes the fluorescence of the fluorophore. The fluorescein structure has two parts confirmed by X-ray analysis, i.e., the benzoic acid moiety as the PET donor and the xanthene ring shown as the fluorophore in previous study [Liu, et al. 2007]. The study showed that only small alterations in absorbance were observed among fluorescein and its derivatives. The dihedral angle () between the benzoic acid moiety and the xanthene ring was almost 90o. It also suggested that there is little ground-state interaction measured by HOMO energy levels between these two parts.

The relationship of HOMO energy levels and () angle of fluorescent compounds predict the fluorescence mechanism of benzoic acid moiety in the compounds. If the HOMO energy level of the benzoic acid moiety is high enough for electron transfer to the excited xanthene ring, the value will be small. In other words, fluorescein derivatives with a high value must have benzoic moieties with low HOMO energy levels. The HOMO energy levels of 3 aminobenzoic acid, 3-benzamidobenzoic acid, 3,4-diaminobenzenecarboxylic acid (DAB-COOH), and benzotriazole-5-carboxylic acid (BT-COOH), which are the benzoic moieties of aminofluorescein, benzamidofluorescein, DAF-2, and DAF-2T, respectively were estimated by semi-empirical (PM3) calculations. DAB-COOH and aminobenzoic acid, which are the benzoic acid moieties of weakly fluorescent fluorescein derivatives, have higher HOMO levels than BT-COOH and amidobenzoic acid. These results were consistent with the PET mechanism.

Further, to confirm this mechanism 9-[2-(3-carboxy)-naphthyl]-6-hydroxy-3H-xanthen-3-one (NX) and 9-[2-(3-carboxy)anthryl]-6-hydroxy-3H-xanthen-3-one (AX) were synthesized [Nagano, et al. 2002]. It is well-known that expanding the size of aromatic conjugates makes their HOMO levels higher, similar with by introducing electron-donating substituents. Thus, naphthoic acid and anthracene-2-carboxylic acid would have higher HOMO levels than benzoic acid, and therefore, the fluorescence properties of NX and AX would differ from those of fluorescein.. The absorbance maximum (Exmax) and emission maximum (Emmax) of NX and AX were closer and not much altered among these fluorescein derivatives. However, the values were greatly altered; NX is highly fluorescent, whereas

EPR spectroscopy at lower frequencies, the L-band (0.4–1.6 GHz) or S-band(1.6–4 GHz), can be used for in vivo EPR imaging. EPR imaging is generally considered to be the most effective technique for noninvasive observation of the spatial distribution of free radicals [Kleschyov, et al. 2007]. Direct EPR detection of endogenous free radicals in biological samples requires that the radical of interest is present at a concentration higher than the practical detection limit (0.1–0.01 μM) and has a relatively long lifetime. Although such requirement can be met at cryogenic temperature, in vivo detection is almost impossible since most radicals do not have a sufficiently long lifetime. Therefore, spin-trapping techniques have been developed in which the spin traps can react with unstable free radicals to form a relatively stable adduct [Berliner, et al. 2004, Davie, et al. 2004]. The formation of the long lived adduct results in accumulation of steady-state free radicals which can be

In principle, the fluorescence properties of fluorescein derivatives are controlled by the photoinduced electron transfer (PET) process from the benzoic acid moiety to the fluorophore, the xanthene ring. PET is a widely accepted alternate mechanism for fluorescence quenching in which electron transfer from the PET donor to the excited fluorophore diminishes the fluorescence of the fluorophore. The fluorescein structure has two parts confirmed by X-ray analysis, i.e., the benzoic acid moiety as the PET donor and the xanthene ring shown as the fluorophore in previous study [Liu, et al. 2007]. The study showed that only small alterations in absorbance were observed among fluorescein and its derivatives. The dihedral angle () between the benzoic acid moiety and the xanthene ring was almost 90o. It also suggested that there is little ground-state interaction measured by

The relationship of HOMO energy levels and () angle of fluorescent compounds predict the fluorescence mechanism of benzoic acid moiety in the compounds. If the HOMO energy level of the benzoic acid moiety is high enough for electron transfer to the excited xanthene ring, the value will be small. In other words, fluorescein derivatives with a high value must have benzoic moieties with low HOMO energy levels. The HOMO energy levels of 3 aminobenzoic acid, 3-benzamidobenzoic acid, 3,4-diaminobenzenecarboxylic acid (DAB-COOH), and benzotriazole-5-carboxylic acid (BT-COOH), which are the benzoic moieties of aminofluorescein, benzamidofluorescein, DAF-2, and DAF-2T, respectively were estimated by semi-empirical (PM3) calculations. DAB-COOH and aminobenzoic acid, which are the benzoic acid moieties of weakly fluorescent fluorescein derivatives, have higher HOMO levels than BT-COOH and amidobenzoic acid. These results were consistent with the PET

Further, to confirm this mechanism 9-[2-(3-carboxy)-naphthyl]-6-hydroxy-3H-xanthen-3-one (NX) and 9-[2-(3-carboxy)anthryl]-6-hydroxy-3H-xanthen-3-one (AX) were synthesized [Nagano, et al. 2002]. It is well-known that expanding the size of aromatic conjugates makes their HOMO levels higher, similar with by introducing electron-donating substituents. Thus, naphthoic acid and anthracene-2-carboxylic acid would have higher HOMO levels than benzoic acid, and therefore, the fluorescence properties of NX and AX would differ from those of fluorescein.. The absorbance maximum (Exmax) and emission maximum (Emmax) of NX and AX were closer and not much altered among these fluorescein derivatives. However, the values were greatly altered; NX is highly fluorescent, whereas

detected readily by EPR spectroscopy.

AX is almost nonfluorescent [Nagano, et al. 2002]. Thus, a small change in the size of conjugated aromatics, namely, from naphthalene to anthracene, causes a great alteration of the fluorescence properties. Authors compared the HOMO levels of the benzoic acid moieties with xanthine ring and naphthoic acid. Napthoic acid moiety is present in highly fluorescent fluorescein and NX. The HOMO levels of benzoic acid moiety were lower than that of the xanthene ring, while the HOMO level of anthracenecarboxylic acid moiety in fluorescent AX, was higher than that of the xanthene. These results were consistent with the idea that a PET process controls the fluorescence properties of fluorescein derivatives and that these properties can be predicted from the HOMO level of the benzoic acid moiety, with a threshold at around -8.9 eV. This in turn provides a basis for developing novel fluorescence probes with fluorescein-derived structure. Still much remains to justify the fluorescent probes in NO bioimaging.

Fig. 8. Representative spin-trapping agents for EPR imaging of NO.

Fe(MGD)2 or Fe(DETC)2 have also been tested for EPR imaging to map the spatial distribution of NO generation in the ischemic myocardium [Kuppusamy, et al. 1996]. Subsequently, Fe(MGD)2 was used in both EPR and MR imaging in rats with sepsis shock [Fujii, et al.1999]. It was reported that Fe(MGD)2 can reduce nitrite to NO at physiological pH, which may cause some detection Inaccuracy as shown in Figure 8, although the rate

Inhibition of Nitric Oxide Synthase Gene Expression:

**Fluorescence imaging of NO**:

similar to that of DAF-2T.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 139

In following section, we will summarize the progress to date on multimodality imaging of NO and NOS expression and address future research directions and obstacles for NO/NOS imaging using biosensors as outlined in Figure 9. For interested readers, basic methods of

2,7-dichlorofluorescin was introduced for NO detection. When it is oxidized by NO, a fluorescent compound (dichlorofluorescein) is formed. Besides a relatively poor detection sensitivity (~ 10 μM of NO), 2,7-dichlorofluorescein also has very low specificity for NO. Later several iron (II) complexes, fluorescently labeled cytochrome c (a hemoprotein that binds NO selectively), cobalt complexes and fluorescent NO cheletropic traps (FNOCTs) were introduced. The iron(II) complex of quinoline pendant cyclam has fluorescence emission at 460 nm, which can be quenched effectively by NO from NO releasing agents. 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) labeled with acridine and Fe(II)-(N-(dithiocarboxy) sarcosine)2 complex (Fe(DTCS)2) (Fig.5), was developed. The addition of an NO-releasing reagent causes a decrease in the

NO detection and imaging are given in Appendix in then end of this chapter.

fluorescence signal due to the irreversible binding of NO to the Fe(II).

 DAFCs are the most successful and extensively investigated agents for NO imaging. In vivo imaging of NO in whole living vertebrate with the DAF compounds was reported, where diaminofluorophore 4-amino-5-methylamino-2′-7′-difluorofluorescein diacetate (DAF-FM-DA) was used to detect NO production sites in the zebrafish Danio rerio. The specificity of the fluorescent signal was confirmed by a decrease in animals exposed to a NO scavenger or a NOS inhibitor, as well as an increase in the presence of a NO donor. Local changes in NO production in response to stressful conditions could also be imaged by this agent, suggesting that DAF-FM-DA can be used to monitor changes in NO production which may have future applications in drug screening and molecular pharmacology. Best example of NO imaging in vivo with the DAF compound in zebrafish is quite transparent. The major limitation of the DAF-2 compound is that it is not specific to NO because it can also react with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles

 Similar to DAF and DAR, rhodamine fluorophore was developed and it was termed "the DAR". To image NO in living cells, DARs should be membrane permeable. Therefore, an ethyl group was introduced into the carboxyl group of DAR-1 (4,5-diamino-N,N,N′,N′ tetra ethyl rhodamine), anticipating that the ethyl ester would be hydrolyzed by cytosolic

 New class of DAQ compounds of DAFC family called diaminocyanines (DACs) were prepared with a tricarbocyanine as the NIR fluorophore and o-phenylenediamine as the NO sensitive fluorescence modulator. It was reported that these compounds can react faster with NO than the DAFs and the reaction efficiency was at least 50 times higher than that of DAF-2. Although it was demonstrated that NO can be imaged in isolated rat

esterases after permeation through the cell membrane [Kojima et al. 2001].

kidneys, in vivo imaging of NO using these compounds has not been reported. A new class of difluoroboradiaza-s-indacene-based agents have been widely used in the detection of NO in cells or tissues [Huang, et al. 2002a]. One of these compounds, 1,3,5,7-tetramethyl-8-(4′-aminophenyl-N-(2′′-amino)-phenzyl)-difluoroboradiaza-sindacene (TMAPABODIPY), was shown to have high photostability, high sensitivity (detection limit is 5 nMof NO), and no pH dependency over a wide pH range (see

constant is quite slow (1–5 M−2 s−1) [Tsuchiya, et al. 2000]. Fe(DETC)2 has been used for imaging vascular NO production in rabbits [Kleschyov, et al. 2000], animal brain injury [Ziaja, et al. 2007], vascular disease [Khoo, et al. 2004], and even in plants [Xu, et al. 2004, Vanin, et al. 2007]. Heme proteins, such as myoglobin, cytochrome c, and catalase, have been used as NO-trapping agents in various biological specimens[Kleschyov, et al. 2000]. Although the EPR signals of heme–NO species can be observed in tissues exposed to elevated levels of NO, it is almost impossible to discriminate between the individual nitrosylated heme proteins due to overlapping of the signal. Generally, the dominant heme– NO EPR signal belongs to the major heme protein expressed in the tissue (e.g., myoglobin in the heart [Konorev, et al. 1996]). The most frequently used heme protein in EPR research is hemoglobin (Hb) [Kosaka, et al. 1994], which can interact with NO to generate several paramagnetic Hb–NO derivatives detectable by EPR spectroscopy [Picknova, et al. 2005]. Different preparations of deoxy-Hb have been used for NO trapping in isolated cells and organelles [Kozlov, et al. 1996].

#### **3.3 NO imaging and fluorescent dyes**

Several fluorescence indicators for direct detection of NO have been generated, which might be useful for in vitro or in vivo NO-imaging. These substances interact with NO to form a fluorescent complex. Several fluorescent dyes have been developed, which can be used for NO bioimaging. However, several of these promising fluorescent dyes have been found not to be very specific such as 2′ ,7′ -dichlorofluorescein (DCF) or to be cytotoxic such as 2,3 diaminonaphthalene (DAN). Of specific mention, iron (II) N-(dithiocarboxyl) sarcosine(Fe(DTCS)2; Fig. 5) trapping agents within 1 h after injection of the spin trap generated stable signal which indicated that the formation of the steady-state free radical had reached equilibrium. The same trapping agent was also tested for NO imaging in lipopolysaccharide-treated mice [Quaresima, et al. 1996]. Subsequently, EPR measurements and EPR-CT (CT denotes computed tomography) imaging with Fe(DTCS)2 were carried out in live mice [Yokoyama, et al. 1997]. The use of Fe(DTCS)2 resulted in a clear EPR-CT image showing high intensity areas in the ventral regions, while other NO-bound iron complexes such as Nmethyl-D-glucamine dithiocarbamate (Fe (MGD)2; Fig. 5) or N,Ndiethyldithiocarbamate (Fe(DETC)2) gave much lower signal-to-noise ratios. Several compounds such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Fig. 5), α-phenyl-N-tertbutylnitrone(PBN), or 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), NO cheletopic traps (NOCTs), aci anions of nitroalkanes, iron (II)–dithiocarbamate(DTC) complex and(DAFs) are fluorescein derivatives. DAFs themselves scarcely fluoresce while DAF-Ts are strong fluorescent compounds. However, the mechanisms accounting for the diminution of fluorescence in DAFs and fluorescence enhancement in DAF-Ts remain unclear.Other fluorescent Fe(DTCS)2 and Cobalt based dyes have been tested in some cell culture systems, but these substances have not been tested in in vivo or in vitro acute brain slice preparations or tissues [von Bohlen, et al. 2003, Zhou, et al. 2009]. Until now, several other fluorescent dyes have been promising candidates for the evaluation of the temporal and spatial aspects of NO generation and distribution even under microscopic observation, including diaminofluoresceins (DAFC), diaminorhodamines (DAR), diaminoanthraquinone (DAQ), fluorescent resonance energy transfer (FRET) and the "fluorescent nitric oxide cheletropic traps" as shown in Table 3 [von Bohlen, et al. 2003, Zhou, et al. 2009].

In following section, we will summarize the progress to date on multimodality imaging of NO and NOS expression and address future research directions and obstacles for NO/NOS imaging using biosensors as outlined in Figure 9. For interested readers, basic methods of NO detection and imaging are given in Appendix in then end of this chapter.

#### **Fluorescence imaging of NO**:

138 Enzyme Inhibition and Bioapplications

constant is quite slow (1–5 M−2 s−1) [Tsuchiya, et al. 2000]. Fe(DETC)2 has been used for imaging vascular NO production in rabbits [Kleschyov, et al. 2000], animal brain injury [Ziaja, et al. 2007], vascular disease [Khoo, et al. 2004], and even in plants [Xu, et al. 2004, Vanin, et al. 2007]. Heme proteins, such as myoglobin, cytochrome c, and catalase, have been used as NO-trapping agents in various biological specimens[Kleschyov, et al. 2000]. Although the EPR signals of heme–NO species can be observed in tissues exposed to elevated levels of NO, it is almost impossible to discriminate between the individual nitrosylated heme proteins due to overlapping of the signal. Generally, the dominant heme– NO EPR signal belongs to the major heme protein expressed in the tissue (e.g., myoglobin in the heart [Konorev, et al. 1996]). The most frequently used heme protein in EPR research is hemoglobin (Hb) [Kosaka, et al. 1994], which can interact with NO to generate several paramagnetic Hb–NO derivatives detectable by EPR spectroscopy [Picknova, et al. 2005]. Different preparations of deoxy-Hb have been used for NO trapping in isolated cells and

Several fluorescence indicators for direct detection of NO have been generated, which might be useful for in vitro or in vivo NO-imaging. These substances interact with NO to form a fluorescent complex. Several fluorescent dyes have been developed, which can be used for NO bioimaging. However, several of these promising fluorescent dyes have been found not

diaminonaphthalene (DAN). Of specific mention, iron (II) N-(dithiocarboxyl) sarcosine(Fe(DTCS)2; Fig. 5) trapping agents within 1 h after injection of the spin trap generated stable signal which indicated that the formation of the steady-state free radical had reached equilibrium. The same trapping agent was also tested for NO imaging in lipopolysaccharide-treated mice [Quaresima, et al. 1996]. Subsequently, EPR measurements and EPR-CT (CT denotes computed tomography) imaging with Fe(DTCS)2 were carried out in live mice [Yokoyama, et al. 1997]. The use of Fe(DTCS)2 resulted in a clear EPR-CT image showing high intensity areas in the ventral regions, while other NO-bound iron complexes such as Nmethyl-D-glucamine dithiocarbamate (Fe (MGD)2; Fig. 5) or N,Ndiethyldithiocarbamate (Fe(DETC)2) gave much lower signal-to-noise ratios. Several compounds such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO; Fig. 5), α-phenyl-N-tertbutylnitrone(PBN), or 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), NO cheletopic traps (NOCTs), aci anions of nitroalkanes, iron (II)–dithiocarbamate(DTC) complex and(DAFs) are fluorescein derivatives. DAFs themselves scarcely fluoresce while DAF-Ts are strong fluorescent compounds. However, the mechanisms accounting for the diminution of fluorescence in DAFs and fluorescence enhancement in DAF-Ts remain unclear.Other fluorescent Fe(DTCS)2 and Cobalt based dyes have been tested in some cell culture systems, but these substances have not been tested in in vivo or in vitro acute brain slice preparations or tissues [von Bohlen, et al. 2003, Zhou, et al. 2009]. Until now, several other fluorescent dyes have been promising candidates for the evaluation of the temporal and spatial aspects of NO generation and distribution even under microscopic observation, including diaminofluoresceins (DAFC), diaminorhodamines (DAR), diaminoanthraquinone (DAQ), fluorescent resonance energy transfer (FRET) and the "fluorescent nitric oxide cheletropic

organelles [Kozlov, et al. 1996].

to be very specific such as 2′

**3.3 NO imaging and fluorescent dyes**

,7′

traps" as shown in Table 3 [von Bohlen, et al. 2003, Zhou, et al. 2009].

2,7-dichlorofluorescin was introduced for NO detection. When it is oxidized by NO, a fluorescent compound (dichlorofluorescein) is formed. Besides a relatively poor detection sensitivity (~ 10 μM of NO), 2,7-dichlorofluorescein also has very low specificity for NO. Later several iron (II) complexes, fluorescently labeled cytochrome c (a hemoprotein that binds NO selectively), cobalt complexes and fluorescent NO cheletropic traps (FNOCTs) were introduced. The iron(II) complex of quinoline pendant cyclam has fluorescence emission at 460 nm, which can be quenched effectively by NO from NO releasing agents. 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) labeled with acridine and Fe(II)-(N-(dithiocarboxy) sarcosine)2 complex (Fe(DTCS)2) (Fig.5), was developed. The addition of an NO-releasing reagent causes a decrease in the fluorescence signal due to the irreversible binding of NO to the Fe(II).

Inhibition of Nitric Oxide Synthase Gene Expression:

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 141

Fig. 9. Imaging NO with diamino-aromatic fluorescent compounds. (A) The general reaction

(B) Fluorescence image of live zebrafish larvae loaded with DAF-FM-DA. Blue arrow, heart; orange arrow, the cleithrum; purple arrow, the notochord; yellow arrow, the caudal fin. (C) Distribution of NO in B16F10 tumors grown in the cranial window in mouse. Left: microangiography using tetramethylrhodamine-dextran; middle: representative

that leads to NO detection by fluorescence with this class of agents.

Figure 9). Subsequently, a similar compound, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8- (3′,4′diaminophenyldifluoroboradiaza-s-indacene (TMDCDABODIPY), was applied for real-time imaging of NO in cells [Huang, et al. 2002b].

Table 3. Properties of fluorescent NO-indicators

Emission (color) ~520 nm (green) ~515 nm(green) >580 nm(red) 460 nm(blue) Excitation ~460 nm ~495 nm ~550 nm ~520 nm Reacts with ROS Generates No -- No

Neurotoxic -- No -- No

 In copper-based fluorescent compounds copper (II) is coordinated with certain fluorophores, the base-catalyzed reaction with NO leads to the reduction of the metal center and the intramolecular nitrosylation of a secondary amine. Based on this mechanism, a series of fluophore coordinated copper (II) complexes were designed for NO imaging [Lim, 2007]. Reduction of the Cu (II) core by NO to Cu(I) with nitrosation of the fluorescent ligand is accompanied by bright fluorescence emission. Any nitrogen/oxygen reactive species or ascorbic acid do not cause any significant fluorescence alteration in Cu(II)-based agents. Cu(II) complexes are quite successful in NO imaging in cell culture but they have not been tested in vivo likely due to the suboptimal emission wavelength, the potential toxicity, and the poor in vivo stability of the Cu(II) complex. Recently, several benzimidazole derivatives have been tested in vivo for NO imaging [Quyang, et al. 2008]. The fluorescent 5′-chloro-2-(2′-hydroxyphenyl)-1Hnaphtho[2,3-d]imidazol and 4-methoxy-2-(1H-naphtho [2,3-d]imidazol-2-yl)phenol can coordinate with Cu(II) to form a nonfluorescent coordination compound, which can detect NO production in inflammation with a turn-on fluorescence. NO production was imaged successfully in both activated murine macrophages and an acute severe hepatic injury (ASHI) mouse model. Although these agents represent the first successful attempt of in vivo fluorescence imaging of NO, the actual imaging was performed ex vivo on frozen tissue slices due to the suboptimal fluorescence emission wavelength as shown in Figure 10. Much further optimization of these Cu(II)-based agents will be needed in the future, in particular fluorescence emission in the NIR range and reduction of the toxicity. Imaging NO/NOS with FRET: FRET agents react with NO covalently a irreversible sensors for NO. Further, some of the organic dyes can accumulate in subcellular membranes and emit fluorescence signals in an NO-independent manner which can cause significant background signal and interfere with the detection of NO. To overcome these limitations, a NO-selective sensor with the heme domain of soluble guanylate cyclase (sGC) was developed [Barker, et al. 1999]. The heme domain of the sGC was labeled with a fluorescent dye and domain changes in the fluorescence intensity of the dye based on the sGC heme domain's characteristic binding of NO. It was found that these sensors have fast, linear, and reversible responses to NO unaffected by other radical species. However, the detection limit is only about 10 μM of

real-time imaging of NO in cells [Huang, et al. 2002b].

Table 3. Properties of fluorescent NO-indicators

NO which makes less useful.

Tested in

neurotoxic tissues

Figure 9). Subsequently, a similar compound, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8- (3′,4′diaminophenyldifluoroboradiaza-s-indacene (TMDCDABODIPY), was applied for

MGD DAF DAR DAQ

No Yes No Yes

Fig. 9. Imaging NO with diamino-aromatic fluorescent compounds. (A) The general reaction that leads to NO detection by fluorescence with this class of agents.

(B) Fluorescence image of live zebrafish larvae loaded with DAF-FM-DA. Blue arrow, heart; orange arrow, the cleithrum; purple arrow, the notochord; yellow arrow, the caudal fin. (C) Distribution of NO in B16F10 tumors grown in the cranial window in mouse. Left: microangiography using tetramethylrhodamine-dextran; middle: representative

Inhibition of Nitric Oxide Synthase Gene Expression:

imaging/detection.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 143

Subsequently, a genetically encoded fluorescent indicator for NO was developed based on FRET, which can reversibly detects NO with high sensitivity (detection limit of 0.1 nM) and visualizes the nanomolar dynamics of NO in single living cells on NO induction [Jares-Erijman, et al. 2003]. However, this approach requires genetic encoding and it generates the biologically active molecule cyclic guanosine monophosphate (cGMP), which can induce other cellular responses. The cGMP molecules can bind to both the NO-associated and the No free probe, resulting in an increase in fluorescence. Therefore, the signal generated is a reflection of the intracellular concentration of cGMP rather than that of NO per se. Recently, the use of confocal based spectral imaging with NO-sensitive FRET reporters enabled NO imaging in the vasculature of intact, isolated perfused mouse lung[St Croix, et al. 2008]. Using calibration spectra, it is possible to unambiguously separate the cross-talk between overlapping donor and acceptor emissions. Overall, FRET is a very powerful technique in cell-based experiments but with only very limited potential applications in animal studies as shown in Figure 11.

**3.4 NO bioimaging by bioluminescence and chemiluminescence imaging**

The major application of chemiluminescence in NO imaging/ detection lies in gas phase or liquid phase measurements[Taha, et al. 2003]. The major chemiluminescence activators for NO imaging include ozone, luminol, and lucigenin. Gas phase chemiluminescence detection of NO is generally based on the reaction of NO with ozone to produce excited-state nitrogen dioxide. When it returns to the normal state, light is emitted at about 600 nm wavelength. Such gas phase detection has been widely used for measuring NO in exhaled breath to detect inflammatory diseases of the lung. Gas phase chemiluminescence detection has very high specificity, as well as extreme sensitivity, which can reach parts per billion (ppb). However, drawbacks include inconvenient instrumentation, high cost, and timeconsuming detection procedure. To overcome these problems, an optimized chemiluminescence detection system for NO was developed. For liquid phase chemiluminescence imaging of NO, currently the luminol/H2O2 detection system is the most useful [Robinson, et al. 1999]. Alkaline luminol can react with NO to generate luminescence. The presence of H2O2 as an activator can enhance the sensitivity by about 20-fold. Two approaches have been used for luminol/H2O2-based imaging of NO. In the first approach, the sample is directly added to the luminol/H2O2 mixture. This strategy can detect very low levels of NO, which is quite useful for visualizing the production of NO from cells or organs, but it lacks NO specificity since luminol can react with many free radical species. In the second strategy, a selective dialysis membrane is placed between the sample and the luminol/H2O2 mixture. This procedure can offer better specificity yet it suffers from poor sensitivity, slow response time, and undesirable NO detection limit (μM level). Besides ozone and luminol, lucigenin is can also be used for NO detection. Lucigenin has been widely used as a chemiluminescent substrate to monitor vascular superoxide formation. In particular, lucigenin at a concentration of 5 μM could be used as an indirect probe to estimate basal vascular NO release. Generally, chemiluminescence is a very sensitive method for NO imaging/detection. However, the disadvantages such as low specificity and time-consuming procedure make chemiluminescence not very useful as an independent technique for NO

Several other imaging techniques have been investigated for NO detection. As an important component of molecular imaging, ultrasound is not inherently suitable for direct NO imaging, although it can be used for evaluating the endothelial function or vasodilation

microfluorography captured 1 h after the loading of DAF-2 in tumors; right: The animals were treated with NOS inhibitor. Color bar shows calibration of the fluorescence intensity with known concentrations of DAF- 2T. (D) Left: fluorescent image of endothelial cells with DAR-4M AM at 10 min after the stimulation. Right: an antagonist of the stimuli strongly abolished the fluorescence intensity. (E) Confocal laser fluorescence microscopy image of cells loaded with DAQ and stimulated to produce NO. Left: bright-field; middle: 488 nm excitation; right: 543 nm excitation. (F) Bright-field and fluorescence images of activated PC12 cells loaded with TMDCDABODIPY in the presence of arginine. Adapted from [Huang, et al. 2007a, 2007b].

Fig. 10. Copper-based fluorescence imaging of NO. (A) CuFL detection of NO produced by iNOS in macrophage cells after different time of incubation. Top: fluorescence images; Bottom, bright-field images. (B) Fluorescence images of excised liver slices harvested from live animals after intravenous injection MNIP-Cu. Left: normal mouse liver; center: ASHI liver; right: Kupffer cell-depleted ASHI liver. Adapted from reference [Quyang, et al. 2008].

microfluorography captured 1 h after the loading of DAF-2 in tumors; right: The animals were treated with NOS inhibitor. Color bar shows calibration of the fluorescence intensity with known concentrations of DAF- 2T. (D) Left: fluorescent image of endothelial cells with DAR-4M AM at 10 min after the stimulation. Right: an antagonist of the stimuli strongly abolished the fluorescence intensity. (E) Confocal laser fluorescence microscopy image of cells loaded with DAQ and stimulated to produce NO. Left: bright-field; middle: 488 nm excitation; right: 543 nm excitation. (F) Bright-field and fluorescence images of activated PC12 cells loaded with TMDCDABODIPY in the presence of arginine. Adapted from

Fig. 10. Copper-based fluorescence imaging of NO. (A) CuFL detection of NO produced by iNOS in macrophage cells after different time of incubation. Top: fluorescence images; Bottom, bright-field images. (B) Fluorescence images of excised liver slices harvested from live animals after intravenous injection MNIP-Cu. Left: normal mouse liver; center: ASHI liver; right: Kupffer cell-depleted ASHI liver. Adapted from reference [Quyang, et al. 2008].

[Huang, et al. 2007a, 2007b].

Subsequently, a genetically encoded fluorescent indicator for NO was developed based on FRET, which can reversibly detects NO with high sensitivity (detection limit of 0.1 nM) and visualizes the nanomolar dynamics of NO in single living cells on NO induction [Jares-Erijman, et al. 2003]. However, this approach requires genetic encoding and it generates the biologically active molecule cyclic guanosine monophosphate (cGMP), which can induce other cellular responses. The cGMP molecules can bind to both the NO-associated and the No free probe, resulting in an increase in fluorescence. Therefore, the signal generated is a reflection of the intracellular concentration of cGMP rather than that of NO per se. Recently, the use of confocal based spectral imaging with NO-sensitive FRET reporters enabled NO imaging in the vasculature of intact, isolated perfused mouse lung[St Croix, et al. 2008]. Using calibration spectra, it is possible to unambiguously separate the cross-talk between overlapping donor and acceptor emissions. Overall, FRET is a very powerful technique in cell-based experiments but with only very limited potential applications in animal studies as shown in Figure 11.

#### **3.4 NO bioimaging by bioluminescence and chemiluminescence imaging**

The major application of chemiluminescence in NO imaging/ detection lies in gas phase or liquid phase measurements[Taha, et al. 2003]. The major chemiluminescence activators for NO imaging include ozone, luminol, and lucigenin. Gas phase chemiluminescence detection of NO is generally based on the reaction of NO with ozone to produce excited-state nitrogen dioxide. When it returns to the normal state, light is emitted at about 600 nm wavelength. Such gas phase detection has been widely used for measuring NO in exhaled breath to detect inflammatory diseases of the lung. Gas phase chemiluminescence detection has very high specificity, as well as extreme sensitivity, which can reach parts per billion (ppb). However, drawbacks include inconvenient instrumentation, high cost, and timeconsuming detection procedure. To overcome these problems, an optimized chemiluminescence detection system for NO was developed. For liquid phase chemiluminescence imaging of NO, currently the luminol/H2O2 detection system is the most useful [Robinson, et al. 1999]. Alkaline luminol can react with NO to generate luminescence. The presence of H2O2 as an activator can enhance the sensitivity by about 20-fold. Two approaches have been used for luminol/H2O2-based imaging of NO. In the first approach, the sample is directly added to the luminol/H2O2 mixture. This strategy can detect very low levels of NO, which is quite useful for visualizing the production of NO from cells or organs, but it lacks NO specificity since luminol can react with many free radical species. In the second strategy, a selective dialysis membrane is placed between the sample and the luminol/H2O2 mixture. This procedure can offer better specificity yet it suffers from poor sensitivity, slow response time, and undesirable NO detection limit (μM level). Besides ozone and luminol, lucigenin is can also be used for NO detection. Lucigenin has been widely used as a chemiluminescent substrate to monitor vascular superoxide formation. In particular, lucigenin at a concentration of 5 μM could be used as an indirect probe to estimate basal vascular NO release. Generally, chemiluminescence is a very sensitive method for NO imaging/detection. However, the disadvantages such as low specificity and time-consuming procedure make chemiluminescence not very useful as an independent technique for NO imaging/detection.

Several other imaging techniques have been investigated for NO detection. As an important component of molecular imaging, ultrasound is not inherently suitable for direct NO imaging, although it can be used for evaluating the endothelial function or vasodilation

Inhibition of Nitric Oxide Synthase Gene Expression:

developed multimodal techniques in their infancy.

significantly help in this aspect [Cai, et al.2007a, 2007b].

**3.5.1 Optical imaging of NOS expression**

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 145

in the same subjects [Cai, et al. 2008a, 2008b]. In the clinical setting, molecular imaging can significantly aid in early lesion detection, patient stratification, and treatment monitoring, which can allow for much earlier diagnosis, earlier treatment, better prognosis, and individualized patient management. To date, the multimodal imaging modalities for visualization of NO include optical imaging (fluorescence and chemiluminescence), electron paramagnetic resonance (EPR) imaging, and MRI. Meanwhile, NOS has been mainly investigated using optical and PET imaging. In following section, we describe recently

Imaging enzyme expression and function is quite challenging. Compared to the vast number of literature reports on NO imaging, much less effort has been directed toward NOS imaging. Briefly, the major modalities for NOS imaging include optical imaging, PET, and some other techniques such as electron microscopy. Historically, the study of NOS physiology was constrained by the lack of suitable probes to detect NOS in living cells or animals. In one pioneering study, a fluorescent iNOS inhibitor, pyrimidine imidazole FITC (PIF), was developed and investigated for microscopy of iNOS in living cells [Panda, et al.2005]. It is an isoform-selective molecule with high affinity to iNOS and the binding is essentially irreversible, which allowed for iNOS imaging in many cell types as well as freshly obtained human lung epithelium. This study indicated that fluorescent probes (e.g., PIF) can be valuable for studying iNOS cell biology and understanding the pathophysiology of diseases that involve dysfunctional iNOS expression. In another report, immunofluorescence staining was performed to evaluate nNOS expression on left inferior alveolar nerve (IAN) injury in ferrets [Davies, et al. 2004]. A possible translocation of nNOS protein from the cell body to the site of nerve injury was proposed. Lastly, some ruthenium(II) and rhenium(I)-diimine wires were reported to be capable of binding to iNOS in vitro to generate NIR fluorescence (710 nm emission), which may potentially be useful as a fluorescent imaging probe for iNOS [Dunn, et al. 2005]. The inflammatory modulating effects of iNOS on laser therapy were studied using bioluminescence imaging (BLI) [Moriyama, et al. 2005]. In this study, the efficiency of different wavelengths laser therapy at different wavelengths in modulating iNOS expression was determined using transgenic animals with a luciferase gene driven by the iNOS gene. On induction of inflammation followed by laser treatment, imaging of iNOS expression was performed at various times by measuring the bioluminescence signal. When compared with the nonirradiated animals, a significant increase in the bioluminescence signal was observed after laser irradiation which demonstrated that iNOS expression can be detected by noninvasive BLI. Comparing the two strategies for NOS imaging with optical techniques, many fluorescently labeled NOS inhibitors have charges which makes them not cell membrane permeable. NOS-controlled luciferase expression appears to be a preferable choice, yet much further optimization will be needed in future studies. Generally speaking, the limited penetration of light in tissue renders optical imaging only suitable for a limited number of scenarios (e.g., superficial regions-of-interest and endoscopy-accessible tissues). Another drawback of optical imaging is that it cannot give accurate quantitative analysis of the imaging result. Combination of quantum dot optical and radionuclide-based imaging techniques (e.g., PET) may

caused by NO [Heiss, et al. 2008]. It is worth noting that ultrasound at different frequencies can stimulate NO production in different contexts such as in endothelial cells, osteoblasts, and vascular dilation. A few other techniques such as time-resolved photoion and photoelectron imaging, Hb spectral alteration imaging, laser induced fluorescence detection (LIF), and calcium amplification imaging have also been studied for NO detection. In particular, LIF imaging was able to detect the NO release in a single cell, which makes it a very powerful tool for studying the kinetics of NO release by neuronal cells during neurotransmission. PET imaging of anesthetized dogs with inhaled radiolabeled 13NO has been performed to study the in vivo kinetics of NO [McCarthy, et al. 1996]. In future, molecular imaging may provide more accurate information on NO in intact living systems. EPR-CT [Yokoyama, et al. 1997], EPR-MRI [Berliner, et al. 2004], and EPRchemiluminescence imaging [Nagano, 2002] have all been attempted and much further research/optimization will be needed before any potential clinical investigation can be in place.

#### **3.5 Multimodal bioimaging of NOS gene expression**

Initially NOS was considered a sole enzyme to produce NO. Nitric oxide synthase (NOS) is also under extensive investigation [Crowell et al. 2003]. NO is biosynthesized as three distinct mammalian NOS isoforms shown in section 1. Neuronal NOS (nNOS or NOS I) and endothelial NOS (eNOS or NOS III) are constitutively expressed in neuronal and endothelial cells, respectively and both are referred to as cNOS. Inducible NOS (iNOS or NOS II) is expressed in cells involved in inflammation, such as macrophages and microglias when stimulated by cytokines and/or endotoxins. Although cNOS and iNOS do not have a significant difference in activity (both function through NO), cNOS expression can be stimulated by Ca2+ whereas iNOS activity is Ca2+-independent. Generally, the NO levels produced by cNOS in stimulated endothelial and neuronal cells are much lower (in nanomolar [nM] range) than those generated by iNOS in macrophages (micromolar [μM] range). Aside from NOS synthesis, recent studies have pointed out that nitrite reduction can also serve as a possible source of biologically relevant NO [Gladwin, et al. 2005]. Excessive production of NO is believed to be responsible for various pathophysiologies, while very low levels of NO are needed to maintain normal physiological conditions. Since the production of NO is closely associated with the expression of NOS, blocking NOS expression is a more convenient approach to inhibit the function of NO than intervention through NO itself.

Molecular imaging refers to the characterization of NO/NOS and measurement of biological processes at the molecular level [Massoud, et al. 2003]. It takes advantage of traditional diagnostic imaging techniques and introduces molecular probes to measure the expression of indicative molecular markers at different stages of diseases. Molecular imaging modalities include molecular magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), optical imaging, targeted ultrasound, single photon emission computed tomography (SPECT), and positron emission tomography (PET). Many hybrid systems that combine two or more of these modalities are already commercially available and certain others are under active development. Molecular imaging can give whole body readout in an intact system at the cost of less workload, inexpansive drug development trial, and provide more statistically relevant results since longitudinal studies can be performed in the same subjects [Cai, et al. 2008a, 2008b]. In the clinical setting, molecular imaging can significantly aid in early lesion detection, patient stratification, and treatment monitoring, which can allow for much earlier diagnosis, earlier treatment, better prognosis, and individualized patient management. To date, the multimodal imaging modalities for visualization of NO include optical imaging (fluorescence and chemiluminescence), electron paramagnetic resonance (EPR) imaging, and MRI. Meanwhile, NOS has been mainly investigated using optical and PET imaging. In following section, we describe recently developed multimodal techniques in their infancy.

#### **3.5.1 Optical imaging of NOS expression**

144 Enzyme Inhibition and Bioapplications

caused by NO [Heiss, et al. 2008]. It is worth noting that ultrasound at different frequencies can stimulate NO production in different contexts such as in endothelial cells, osteoblasts, and vascular dilation. A few other techniques such as time-resolved photoion and photoelectron imaging, Hb spectral alteration imaging, laser induced fluorescence detection (LIF), and calcium amplification imaging have also been studied for NO detection. In particular, LIF imaging was able to detect the NO release in a single cell, which makes it a very powerful tool for studying the kinetics of NO release by neuronal cells during neurotransmission. PET imaging of anesthetized dogs with inhaled radiolabeled 13NO has been performed to study the in vivo kinetics of NO [McCarthy, et al. 1996]. In future, molecular imaging may provide more accurate information on NO in intact living systems. EPR-CT [Yokoyama, et al. 1997], EPR-MRI [Berliner, et al. 2004], and EPRchemiluminescence imaging [Nagano, 2002] have all been attempted and much further research/optimization will be needed before any potential clinical investigation can be in

Initially NOS was considered a sole enzyme to produce NO. Nitric oxide synthase (NOS) is also under extensive investigation [Crowell et al. 2003]. NO is biosynthesized as three distinct mammalian NOS isoforms shown in section 1. Neuronal NOS (nNOS or NOS I) and endothelial NOS (eNOS or NOS III) are constitutively expressed in neuronal and endothelial cells, respectively and both are referred to as cNOS. Inducible NOS (iNOS or NOS II) is expressed in cells involved in inflammation, such as macrophages and microglias when stimulated by cytokines and/or endotoxins. Although cNOS and iNOS do not have a significant difference in activity (both function through NO), cNOS expression can be stimulated by Ca2+ whereas iNOS activity is Ca2+-independent. Generally, the NO levels produced by cNOS in stimulated endothelial and neuronal cells are much lower (in nanomolar [nM] range) than those generated by iNOS in macrophages (micromolar [μM] range). Aside from NOS synthesis, recent studies have pointed out that nitrite reduction can also serve as a possible source of biologically relevant NO [Gladwin, et al. 2005]. Excessive production of NO is believed to be responsible for various pathophysiologies, while very low levels of NO are needed to maintain normal physiological conditions. Since the production of NO is closely associated with the expression of NOS, blocking NOS expression is a more convenient approach to inhibit the function of NO than intervention

Molecular imaging refers to the characterization of NO/NOS and measurement of biological processes at the molecular level [Massoud, et al. 2003]. It takes advantage of traditional diagnostic imaging techniques and introduces molecular probes to measure the expression of indicative molecular markers at different stages of diseases. Molecular imaging modalities include molecular magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), optical imaging, targeted ultrasound, single photon emission computed tomography (SPECT), and positron emission tomography (PET). Many hybrid systems that combine two or more of these modalities are already commercially available and certain others are under active development. Molecular imaging can give whole body readout in an intact system at the cost of less workload, inexpansive drug development trial, and provide more statistically relevant results since longitudinal studies can be performed

place.

through NO itself.

**3.5 Multimodal bioimaging of NOS gene expression**

Imaging enzyme expression and function is quite challenging. Compared to the vast number of literature reports on NO imaging, much less effort has been directed toward NOS imaging. Briefly, the major modalities for NOS imaging include optical imaging, PET, and some other techniques such as electron microscopy. Historically, the study of NOS physiology was constrained by the lack of suitable probes to detect NOS in living cells or animals. In one pioneering study, a fluorescent iNOS inhibitor, pyrimidine imidazole FITC (PIF), was developed and investigated for microscopy of iNOS in living cells [Panda, et al.2005]. It is an isoform-selective molecule with high affinity to iNOS and the binding is essentially irreversible, which allowed for iNOS imaging in many cell types as well as freshly obtained human lung epithelium. This study indicated that fluorescent probes (e.g., PIF) can be valuable for studying iNOS cell biology and understanding the pathophysiology of diseases that involve dysfunctional iNOS expression. In another report, immunofluorescence staining was performed to evaluate nNOS expression on left inferior alveolar nerve (IAN) injury in ferrets [Davies, et al. 2004]. A possible translocation of nNOS protein from the cell body to the site of nerve injury was proposed. Lastly, some ruthenium(II) and rhenium(I)-diimine wires were reported to be capable of binding to iNOS in vitro to generate NIR fluorescence (710 nm emission), which may potentially be useful as a fluorescent imaging probe for iNOS [Dunn, et al. 2005]. The inflammatory modulating effects of iNOS on laser therapy were studied using bioluminescence imaging (BLI) [Moriyama, et al. 2005]. In this study, the efficiency of different wavelengths laser therapy at different wavelengths in modulating iNOS expression was determined using transgenic animals with a luciferase gene driven by the iNOS gene. On induction of inflammation followed by laser treatment, imaging of iNOS expression was performed at various times by measuring the bioluminescence signal. When compared with the nonirradiated animals, a significant increase in the bioluminescence signal was observed after laser irradiation which demonstrated that iNOS expression can be detected by noninvasive BLI. Comparing the two strategies for NOS imaging with optical techniques, many fluorescently labeled NOS inhibitors have charges which makes them not cell membrane permeable. NOS-controlled luciferase expression appears to be a preferable choice, yet much further optimization will be needed in future studies. Generally speaking, the limited penetration of light in tissue renders optical imaging only suitable for a limited number of scenarios (e.g., superficial regions-of-interest and endoscopy-accessible tissues). Another drawback of optical imaging is that it cannot give accurate quantitative analysis of the imaging result. Combination of quantum dot optical and radionuclide-based imaging techniques (e.g., PET) may significantly help in this aspect [Cai, et al.2007a, 2007b].

Inhibition of Nitric Oxide Synthase Gene Expression:

al. 2009]

et al. 2009]

**4. Biological applications of NO imaging**

fluctuations during a MRI scanning of NO-related diseases.

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 147

Fig. 11. (on left) Imaging NO with FRET. (A) Basic principles of FRET between CFP and YFP. CFP: cyan fluorescent protein; YFP: yellow fluorescent protein. (B) Pseudocolor images of the CFP/YFP emission ratio of the cell before (1570 s) and after addition of NOC-7, an NO-donating reagent that releases 2 mol of NO per mole. Adapted from reference [Hong et

Fig. 12. (on right) Several NOS inhibiters and an antisense oligonucleotide for iNOS mRNA have been radiolabeled for PET imaging of NOS expression.Adapted from reference [Hong,

Still development and applications of fluorescent agents in cell imaging and NO sensitive MRI contrast agents for in vivo physiological fMRI are in infancy. MRI imaging of NO based on nitrite concentration can detect physiological changes such as cerebral blood volume change in nonischemic spontaneously hypertensive rats [Caramia et al. 1998], increase in tumor blood flow and partial oxygen pressure after NO donor administration [Jordan et al. 2000], NO production alteration in humans after a 30-min exposure to a 1.5 T magnetic field [Sirmatel, et al. 2007]. Interestingly, high magnetic strength can cause NO concentration

#### **3.5.2 PET imaging of NOS expression**

Small animal PET scanners have very good spatial resolution (1 mm) and they are also becoming increasingly widely available. PET does not have a tissue penetration issue, it is highly quantitative, and it can be used for both diagnosis and treatment monitoring. To date, PET imaging of NOS expression has been focused on the clarification of nNOS function in neurological diseases [Pomper, et al. 2000, Zhang, et al. 1997]. PET imaging of nNOS was initially investigated with a 11C-labeled nNOS inhibitor, S-methyl-Lthiocitrulline (MTICU) (FIG 11, 12) [Zhang, et al. 1997]. Biodistribution of 11C-MTICU gave a brain-to-blood ratio of 1:2 at 30 min postinjection. Uptake in the cerebellum (high nNOS expression) was 20% higher than that of the cortex or the brain stem (low nNOS expression). Blockage using 1 mg/kg of MTICU was able to reduce the uptake in the cerebellum and the cortex, but not in the brain stem. Study demonstrated the potential of 11CMTICU as a potentially useful tracer in determining nNOS levels in vivo. In another report, two nNOS-selective PET tracers, AR-R 17443 [N-(4-(2-(phenylmethyl) (methyl) amino)ethyl)phenyl)-2-thiophenecarboximidamide)] and AR-R 18512 [(N(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-yl)-2 thiophenecarboximidamide)] were synthesized and evaluated in rodents and primates [Pomper et al. 2000]. Another strategy has been employed for iNOS imaging using antisense oligonucleotide with hybridization properties for iNOS mRNA using RT-PCR. The oligonucleotide was then labeled with 18F (FIG 12) [de Varies, et al. 2004]. Cellular uptake or efflux showed no selectivity for iNOS expressing cells. Thus PET imaging of NOS expression has not been very successful which is likely due to several reasons. First, the expression of NOS is in the cytoplasm. Although the radiolabeled NOS inhibitors usually have high affinity for NOS, they may not permeate through cell membrane efficiently which dramatically affects the tracer uptake. Second, the specificity of these NOS inhibitors may not be high enough for imaging applications; many of them can undergo nonspecific adsorption to other proteins such as albumin. Third, the stability of those tracers in vivo is a major concern. Uptake in the tumor or other targeted organs may not truly reflect the distribution of the intact tracer. Lastly, these NOS inhibitors are small molecules. Although replacing the 12CH3 with 11CH3 will not change the chemical structure, in many cases this cannot be achieved. Recently, a novel NOS inhibitor design strategy, termed the "anchored plasticity approach," may give rise to novel NOS inhibitors for PET imaging applications in the future [Garcin et al. 2008].

#### **3.5.3 Imaging NOS expression with other techniques**

Besides optical and PET imaging, other techniques have been explored for NOS-related research, mainly electron microscopy and immunohistochemistry [Heinrich, et al. 2005, Lajoix, et al. 2006]. Electron microscopy was applied to visualize the subcellular localization of NOS in different cell organelles using gold particles conjugated with anti-NOS antibodies, which gave exquisite distribution information of NOS in cells. Immunohistochemistry also uses anti-NOS antibodies, easier to perform than electron microscopy yet the resolution is not as high. Both approaches are invasive and not suitable for in vivo imaging; however, the experimental findings are typically quite reliable and thus they can serve as ex vivo validation for in vivo imaging studies.

Small animal PET scanners have very good spatial resolution (1 mm) and they are also becoming increasingly widely available. PET does not have a tissue penetration issue, it is highly quantitative, and it can be used for both diagnosis and treatment monitoring. To date, PET imaging of NOS expression has been focused on the clarification of nNOS function in neurological diseases [Pomper, et al. 2000, Zhang, et al. 1997]. PET imaging of nNOS was initially investigated with a 11C-labeled nNOS inhibitor, S-methyl-Lthiocitrulline (MTICU) (FIG 11, 12) [Zhang, et al. 1997]. Biodistribution of 11C-MTICU gave a brain-to-blood ratio of 1:2 at 30 min postinjection. Uptake in the cerebellum (high nNOS expression) was 20% higher than that of the cortex or the brain stem (low nNOS expression). Blockage using 1 mg/kg of MTICU was able to reduce the uptake in the cerebellum and the cortex, but not in the brain stem. Study demonstrated the potential of 11CMTICU as a potentially useful tracer in determining nNOS levels in vivo. In another report, two nNOS-selective PET tracers, AR-R 17443 [N-(4-(2-(phenylmethyl) (methyl) amino)ethyl)phenyl)-2-thiophenecarboximidamide)] and AR-R 18512 [(N(2-methyl-1,2,3,4-tetrahydroisoquinoline-7-yl)-2 thiophenecarboximidamide)] were synthesized and evaluated in rodents and primates [Pomper et al. 2000]. Another strategy has been employed for iNOS imaging using antisense oligonucleotide with hybridization properties for iNOS mRNA using RT-PCR. The oligonucleotide was then labeled with 18F (FIG 12) [de Varies, et al. 2004]. Cellular uptake or efflux showed no selectivity for iNOS expressing cells. Thus PET imaging of NOS expression has not been very successful which is likely due to several reasons. First, the expression of NOS is in the cytoplasm. Although the radiolabeled NOS inhibitors usually have high affinity for NOS, they may not permeate through cell membrane efficiently which dramatically affects the tracer uptake. Second, the specificity of these NOS inhibitors may not be high enough for imaging applications; many of them can undergo nonspecific adsorption to other proteins such as albumin. Third, the stability of those tracers in vivo is a major concern. Uptake in the tumor or other targeted organs may not truly reflect the distribution of the intact tracer. Lastly, these NOS inhibitors are small molecules. Although replacing the 12CH3 with 11CH3 will not change the chemical structure, in many cases this cannot be achieved. Recently, a novel NOS inhibitor design strategy, termed the "anchored plasticity approach," may give rise to novel NOS inhibitors for PET imaging applications in the

**3.5.2 PET imaging of NOS expression**

future [Garcin et al. 2008].

validation for in vivo imaging studies.

**3.5.3 Imaging NOS expression with other techniques**

Besides optical and PET imaging, other techniques have been explored for NOS-related research, mainly electron microscopy and immunohistochemistry [Heinrich, et al. 2005, Lajoix, et al. 2006]. Electron microscopy was applied to visualize the subcellular localization of NOS in different cell organelles using gold particles conjugated with anti-NOS antibodies, which gave exquisite distribution information of NOS in cells. Immunohistochemistry also uses anti-NOS antibodies, easier to perform than electron microscopy yet the resolution is not as high. Both approaches are invasive and not suitable for in vivo imaging; however, the experimental findings are typically quite reliable and thus they can serve as ex vivo

Fig. 11. (on left) Imaging NO with FRET. (A) Basic principles of FRET between CFP and YFP. CFP: cyan fluorescent protein; YFP: yellow fluorescent protein. (B) Pseudocolor images of the CFP/YFP emission ratio of the cell before (1570 s) and after addition of NOC-7, an NO-donating reagent that releases 2 mol of NO per mole. Adapted from reference [Hong et al. 2009]

Fig. 12. (on right) Several NOS inhibiters and an antisense oligonucleotide for iNOS mRNA have been radiolabeled for PET imaging of NOS expression.Adapted from reference [Hong, et al. 2009]

#### **4. Biological applications of NO imaging**

Still development and applications of fluorescent agents in cell imaging and NO sensitive MRI contrast agents for in vivo physiological fMRI are in infancy. MRI imaging of NO based on nitrite concentration can detect physiological changes such as cerebral blood volume change in nonischemic spontaneously hypertensive rats [Caramia et al. 1998], increase in tumor blood flow and partial oxygen pressure after NO donor administration [Jordan et al. 2000], NO production alteration in humans after a 30-min exposure to a 1.5 T magnetic field [Sirmatel, et al. 2007]. Interestingly, high magnetic strength can cause NO concentration fluctuations during a MRI scanning of NO-related diseases.

Inhibition of Nitric Oxide Synthase Gene Expression:

\*NO imaging [Kojima, et al. 2001, Kojima, et al. 2001].

lungs, heart, and liver [Kuppusamy, et al. 2001].

**4.4 Calcium-imaging and NO-imaging**

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 149

biomedical applications in molecular imaging [Liu, et al. 2007]. In other study, the clinical detection of evolving acute tubular necrosis (ATN) and differentiating it from other causes of renal failure was performed. Herein, sodium magnetic resonance imaging (23Na MRI) was applied to study the early alteration in renal sodium distribution in rat kidneys 6 h after the induction of ATN combined with inhibition of nitric oxide and prostaglandin synthesis. Hence, 23Na MRI non-invasively quantified changes in the corticomedullary sodium gradient in the ATN kidney during morphologic tubular injury [Maril, et al. 2006]. Other newer application of 19F NMR spin-trapping technique for in vivo \*NO detection was employed to elucidate the significance of \*NO availability in animal models of hypertension. In vivo \*NO-induced conversion of the hydroxylamine from fluorinated nitrosyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) was evidenced in hypertensive ISIAH and OXYS rat strains [Bobko, et al. 2005]. The NMR detected positive levels of nitrite/nitrate for *in vivo* evaluation of \*NO production and provided the basis for in vivo

The cardiovascular ischemia in tissues generates the nitric oxide (NO). It is an enzymeindependent mechanism of NO generation and more pathogenesis. So, the NO formation in mice after cardiopulmonary arrest could be imaged. Real-time measurement of NO generation was performed by detection of naturally generated NO-heme complexes in tissues using L-band electron paramagnetic resonance (EPR) spectroscopy. Measurements of NO generation were imaged on the intact animal at the levels of the head, thorax, and abdomen within 3 hours [Ueno, et al. 2002]. Very recently, nitric oxide was recognized as biomarker of vascular dysfunction in renal, clitoris and penile sex organs to detect the physiology [Ockalli, et al. 1999, Kakiailatu, 2000, Burnett, 2002, Barouch, et al. 2002, Hillebrandt, et al. 2009, Ciplak, et al. 2009, Gragasin, et al. 2004, Richards, et al. 2003]. The NO complexes were found to have maximum levels in lung, heart, and liver by threedimensional spatial mapping of the NO complex in the intact animal subjected to cardiopulmonary arrest. The images also confirmed the maximum NO formation in the

The combined real-time bioimaging of calcium and nitric oxide with continuous electrophysiological intracellular recording has gained enormous interest. For intracellular monitoring by calcium imaging in cells, Fura-2 is a calcium-sensitive fluorescent probe. Using Fura-2, intracellular free calcium ion concentration ([Ca2+]i) of single neurons was measured [Richards, et al. 2003]. Calcium-imaging with Fura-2 was used to monitor and modulate the intracellular Ca2+ evoked by NMDA and AMPA in cultured hippocampal pyramidal cells [Keelan, et al. 1999] Calcium and NO play role in excitotoxic mitochondrial depolarization in the hippocampus [Berkels, et al. 2000]. The combination of NO bioimaging with calcium imaging may distinguish the temporal rise in NO and calcium to suggest that NO is being produced as a consequence of Ca2+ enhancement and Ca2+ induced NO Synthase activity). Recently, imaging intracellular calcium imaging was achieved together with NO imaging by using two monochromators DAF-2 and Fura-2 AM as excitation light source. The DAF-2 excited at 485 nm and Fura-2 AM excited at 340 nm [Berkels, et al. 2000]. The emission maximum of fura-2 is about 510 nm different while the emission maximum of DAF-2 is about 515 nm. By using dichroic or multichroic mirrors calcium and NO levels can

The major advantages of MRI include high resolution, good soft tissue contrast, wide availability, sufficiently large field-of-view, and the ability to provide both functional and anatomical information. Nitric oxide bioimaging has emerged as technique to visualize the intracellular metabolism based on redox reactions, mitochondrial oxidation and nitric oxide synthase enzyme NOSi [Anbar, 2000, Zhao, et al. 2009]. Recently, nitric oxide was invented as active mechanism to play role in vascular physiology in lungs, kidney, heart, brain, sex organs. Endothelial cell, smooth muscle cells, oxygenation and perfusion, hypoxia, ischemia are the possible targets of nitric oxide biotransformation. These physiological changes are likely to be visualized by nitric oxide bioimaging in real time manner and reviewed in the following sections.

#### **4.1 Cardiovascular and endothelial cell injury**

DAF-FM is useful tool to visualize the temporal and spatial distribution of intracellular NO. Endogenous ATP plays a central role in HTS-induced NOS in endothelial cell injury. Endothelial cNOS, a calmodulin-dependent enzyme is critical for vascular homeostasis and makes detectable basal level of NO production at low extracellular Ca2+. cNOS expression can be stimulated by Ca2+ while iNOS activity is Ca2+-independent. Actin microfilaments in primary artery endothelial cells (PAEC) regulates L-arginine transport and this regulation can affect NO production by PAEC, DAR-4M may be useful for bioimaging of samples that have strong autofluorescence [Lai, et al. 1994, Kojima, et al. 2001]

#### **4.2 Apoptosis in cells**

Apoptosis plays a key role in many pathological circumstances, such as neurodegenerative diseases. In these processes, the involvement of nitric oxide (NO) has been well established, and the ability of NO to exert cellular damage due to its reactive oxidative properties is perhaps the primary neurotoxic mechanism. Emerging evidences indicate that apoptosis contributes to neuronal death to explain neurodegeneration [Fujii et al. 1997]. The major biochemical change in activation of the cystein protease (caspase-3) was reported to execute apoptosis in the central nervous system disorders such as stroke, spinal cord trauma, head injury and Alzheimer's disease [Green,1998, Nagata,1997 ]. Although numerous techniques have been described to evaluate apoptosis, these approaches involve invasive techniques and cannot provide detailed information about apoptosis *in vivo* [Anbar, 2000].

#### **4.3 Renal and cardiovascular imaging**

A new approach of irreversible responsive PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) for MRI contrast agents constituted a new type of agent for molecular imaging. A novel PARACEST MRI contrast agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid)-orthoaminoanilide (Yb-DO3A-oAA), was developed to image nitric oxide (NO). The agent exhibited two CEST effects at -11 ppm and +8 ppm chemical shifts, which were assigned to chemical exchange from amide and amine functional groups, respectively. This responsive PARACEST MRI contrast agent indicated the change in the presence of NO and O(2), which caused an irreversible disappearance of both PARACEST effects from MR images. This report highlighted the advantages of irreversible MRI contrast agents and demonstrated that large changes in PARACEST can be used to in vivo

The major advantages of MRI include high resolution, good soft tissue contrast, wide availability, sufficiently large field-of-view, and the ability to provide both functional and anatomical information. Nitric oxide bioimaging has emerged as technique to visualize the intracellular metabolism based on redox reactions, mitochondrial oxidation and nitric oxide synthase enzyme NOSi [Anbar, 2000, Zhao, et al. 2009]. Recently, nitric oxide was invented as active mechanism to play role in vascular physiology in lungs, kidney, heart, brain, sex organs. Endothelial cell, smooth muscle cells, oxygenation and perfusion, hypoxia, ischemia are the possible targets of nitric oxide biotransformation. These physiological changes are likely to be visualized by nitric oxide bioimaging in real time manner and reviewed in the

DAF-FM is useful tool to visualize the temporal and spatial distribution of intracellular NO. Endogenous ATP plays a central role in HTS-induced NOS in endothelial cell injury. Endothelial cNOS, a calmodulin-dependent enzyme is critical for vascular homeostasis and makes detectable basal level of NO production at low extracellular Ca2+. cNOS expression can be stimulated by Ca2+ while iNOS activity is Ca2+-independent. Actin microfilaments in primary artery endothelial cells (PAEC) regulates L-arginine transport and this regulation can affect NO production by PAEC, DAR-4M may be useful for bioimaging of samples that

Apoptosis plays a key role in many pathological circumstances, such as neurodegenerative diseases. In these processes, the involvement of nitric oxide (NO) has been well established, and the ability of NO to exert cellular damage due to its reactive oxidative properties is perhaps the primary neurotoxic mechanism. Emerging evidences indicate that apoptosis contributes to neuronal death to explain neurodegeneration [Fujii et al. 1997]. The major biochemical change in activation of the cystein protease (caspase-3) was reported to execute apoptosis in the central nervous system disorders such as stroke, spinal cord trauma, head injury and Alzheimer's disease [Green,1998, Nagata,1997 ]. Although numerous techniques have been described to evaluate apoptosis, these approaches involve invasive techniques

A new approach of irreversible responsive PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) for MRI contrast agents constituted a new type of agent for molecular imaging. A novel PARACEST MRI contrast agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid)-orthoaminoanilide (Yb-DO3A-oAA), was developed to image nitric oxide (NO). The agent exhibited two CEST effects at -11 ppm and +8 ppm chemical shifts, which were assigned to chemical exchange from amide and amine functional groups, respectively. This responsive PARACEST MRI contrast agent indicated the change in the presence of NO and O(2), which caused an irreversible disappearance of both PARACEST effects from MR images. This report highlighted the advantages of irreversible MRI contrast agents and demonstrated that large changes in PARACEST can be used to in vivo

and cannot provide detailed information about apoptosis *in vivo* [Anbar, 2000].

following sections.

**4.2 Apoptosis in cells**

**4.3 Renal and cardiovascular imaging**

**4.1 Cardiovascular and endothelial cell injury**

have strong autofluorescence [Lai, et al. 1994, Kojima, et al. 2001]

biomedical applications in molecular imaging [Liu, et al. 2007]. In other study, the clinical detection of evolving acute tubular necrosis (ATN) and differentiating it from other causes of renal failure was performed. Herein, sodium magnetic resonance imaging (23Na MRI) was applied to study the early alteration in renal sodium distribution in rat kidneys 6 h after the induction of ATN combined with inhibition of nitric oxide and prostaglandin synthesis. Hence, 23Na MRI non-invasively quantified changes in the corticomedullary sodium gradient in the ATN kidney during morphologic tubular injury [Maril, et al. 2006]. Other newer application of 19F NMR spin-trapping technique for in vivo \*NO detection was employed to elucidate the significance of \*NO availability in animal models of hypertension. In vivo \*NO-induced conversion of the hydroxylamine from fluorinated nitrosyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) was evidenced in hypertensive ISIAH and OXYS rat strains [Bobko, et al. 2005]. The NMR detected positive levels of nitrite/nitrate for *in vivo* evaluation of \*NO production and provided the basis for in vivo \*NO imaging [Kojima, et al. 2001, Kojima, et al. 2001].

The cardiovascular ischemia in tissues generates the nitric oxide (NO). It is an enzymeindependent mechanism of NO generation and more pathogenesis. So, the NO formation in mice after cardiopulmonary arrest could be imaged. Real-time measurement of NO generation was performed by detection of naturally generated NO-heme complexes in tissues using L-band electron paramagnetic resonance (EPR) spectroscopy. Measurements of NO generation were imaged on the intact animal at the levels of the head, thorax, and abdomen within 3 hours [Ueno, et al. 2002]. Very recently, nitric oxide was recognized as biomarker of vascular dysfunction in renal, clitoris and penile sex organs to detect the physiology [Ockalli, et al. 1999, Kakiailatu, 2000, Burnett, 2002, Barouch, et al. 2002, Hillebrandt, et al. 2009, Ciplak, et al. 2009, Gragasin, et al. 2004, Richards, et al. 2003]. The NO complexes were found to have maximum levels in lung, heart, and liver by threedimensional spatial mapping of the NO complex in the intact animal subjected to cardiopulmonary arrest. The images also confirmed the maximum NO formation in the lungs, heart, and liver [Kuppusamy, et al. 2001].

#### **4.4 Calcium-imaging and NO-imaging**

The combined real-time bioimaging of calcium and nitric oxide with continuous electrophysiological intracellular recording has gained enormous interest. For intracellular monitoring by calcium imaging in cells, Fura-2 is a calcium-sensitive fluorescent probe. Using Fura-2, intracellular free calcium ion concentration ([Ca2+]i) of single neurons was measured [Richards, et al. 2003]. Calcium-imaging with Fura-2 was used to monitor and modulate the intracellular Ca2+ evoked by NMDA and AMPA in cultured hippocampal pyramidal cells [Keelan, et al. 1999] Calcium and NO play role in excitotoxic mitochondrial depolarization in the hippocampus [Berkels, et al. 2000]. The combination of NO bioimaging with calcium imaging may distinguish the temporal rise in NO and calcium to suggest that NO is being produced as a consequence of Ca2+ enhancement and Ca2+ induced NO Synthase activity). Recently, imaging intracellular calcium imaging was achieved together with NO imaging by using two monochromators DAF-2 and Fura-2 AM as excitation light source. The DAF-2 excited at 485 nm and Fura-2 AM excited at 340 nm [Berkels, et al. 2000]. The emission maximum of fura-2 is about 510 nm different while the emission maximum of DAF-2 is about 515 nm. By using dichroic or multichroic mirrors calcium and NO levels can

Inhibition of Nitric Oxide Synthase Gene Expression:

multichroic mirrors in synaptic plasticity.

**4.7 Alveolar cells**

**4.8 Endothelial cells**

production via the O2-

2002].

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 151

neuronal processes with continuous monitoring the calcium and NO by dichroic or

Human alveolar macrophages and alveolar epithelium is the site of nitric oxide synthase-2 (NOS-2) messenger ribonucleic acid (mRNA) and protein expression in alveolar wall in lung of patients with pulmonary malignancies. In the process of NO production, proinflammatory cytokines interleukin (IL)-1β, tumour necrosis factor (TNF)-, interferon (IFN)- or lipopolysaccharide (LPS) play significant role to induce the effect of human surfactant protein-A (SP-A) on IFN--mediated NOS-2 mRNA expression [Pechkovsky, et al.

Kojima et al. 2001, reported NO bioimaging in cultured bovine aortic endothelial cells. The cultured bovine aortic endothelial cells were incubated with DAF-FM DA for 1 h for dye loading. After stimulation with bradykinin, the fluorescence intensity in the cells increased and that in the cytosol increased more than that in the nucleus. NO is produced in the cytosol, where NOS exists [von Bohlan, et al. 2002]. This observation implies that little of the produced NO diffuses into the nucleus or if it does diffuse into the nucleus little of it is oxidized there. The augmentation of the fluorescence intensity was suppressed by an NOS inhibitor. In conclusion, DAF-FM is a useful tool for visualizing the temporal and spatial distribution of intracellular NO. Kimura et al.2001, examined the effects of acute glucose overload on the cellular productivity of NO in bovine aortic endothelial cells (BAEC) using DAF-2. ATP induced an increase in NO production, assessed by DAF-2, mainly due to Ca2+ entry as shown in Figure 13. In contrast, the ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload. The results indicate that glucose overload impairs NO

examined in detail the mechanism by which mechanical stress induces NOS in endothelium. Hypotonic stress (HTS) induced ATP release, which evoked Ca2+ transients in BAEC. HTS also induced NOS, assessed by DAF-2 fluorescence. The results obtained indicate that endogenous ATP plays a central role in HTS-induced NOS in BAEC. Broillet et al. 2001, reported that Ca2+, Mg2+, or incident light promoted the reaction of DAF-2 and NO. However, Nagano et al. 2002, probed that the enhancement of fluorescence intensity was caused not by increasing the reaction rate between DAF-2 and NO but by promotion of NOreleasing rate of NO donors. Endothelial NOS, a Ca2+/calmodulin-dependent enzyme, is critical for vascular homeostasis [Suzuki, et al. 2002]. To determine the signaling pathway for endogenous eNOS, Lin et al. 2000, directly measured NO production in bovine pulmonary artery endothelial cells (PAEC). Endothelial cells grown in a monolayer produce very low levels of NO which are below the detection range of traditional assays. Therefore, they employed DAF-2 DA to measure NO production in real-time. PAEC had detectable basal NO production which increased slightly after thapsigargin treatment in the presence of low extracellular Ca2+. In contrast, a large increase in NO production was detected in the presence of 4 mM extracellular Ca2+, suggesting that capacitative Ca2+ entry regulates wildtype eNOS activity. Zharikov et al. 2001, investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/NO pathway in pulmonary artery

be measured simultaneously [Pittner, et al. 2003]. Other example of monochromator complex is DAQ and DAR-4M AM. These are calcium sensitive non-neurotoxic dyes with unique fluorescent patterns of DAQ (excitation 520 nm; emission 580 nm) and DAR-4M AM (excitation 550 nm; emission 580 nm). These can also combine with the calciumsensitive dye Fura-2 as potential tools in fluorescence microscopy.

#### **4.5 Kupffer cell NO bioimaging**

A new mechanism was reported in nonparenchymal cells based on Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, was upregulated in vitro and in vivo by nitric oxide (NO) downregulation of BNIP3 gene expression as one mechanism of hepatocyte cell death and liver damage. Authors proposed that inflammatory stresses can lead to the modulation of BNIP3 [Metukuri, et al. 2009].

#### **4.6 Combining electrophysiology and NO-imaging**

The calcium-imaging with electrophysiological recording was excitement in the hippocampus imaging art because of calcium accumulation in hippocampus pyramidal cells during synaptic activation [Regehr, et al. 1989]. Now a day, calcium-imaging combined with electrophysiological stimulation and recording has emerged as present state of art of calcium-signaling and intracellular free calcium ion concentration [Ca2+]i of single neurone. Moreover, little is understood how neuronal signals stimulate NO production. Potential applications of fluorescent NO indicators in bioimaging are to monitor NO in response to pharmacological manipulation using NO-donors, NOS substrate, and NOS or NOS isoform inhibitors as enhancers of NO production in vivo and in vitro [Moore, et al. 1997, Southan, et al. 1996]*.* Other emerging techniques of bioimaging by NO inhibitor are targeting the differential cofactor requirements of the various isoforms; pharmacological agents targeting the differential substrate requirements of cell expression of various isoforms of the NOS [Salerno, et al. 2002]. However, this approach suffers due to lack a marked isoform selectivity or direct affecting the arginine transport into cells, like *N*G-methyl-L-arginine (L-NMA). Other example of NOS inhibitors such as 7-nitroindazole (7-NI), might have unexpected side-effects in neuronal tissues, since 7-NI interferes with monoamine oxidase [Castagnoli, et al. 1999, Desvignes, et al. 1999]. However, selective NOS-inhibitors together with fluorescent NO-imaging appear as useful tools for the investigation of the biological functions of the different NOS-isoforms in neuronal tissues [Chen, et al. 2001, Brown, et al. 1999]. The combined NO-imaging with in vitro electrophysiology methods visualize the NO generation and NO distribution with localization of NO induced by neurotransmitter stimulations, e.g., NO formation after neuronal stimulation with NMDA, in normal synaptic transmission, in paired-pulse facilitation, in long-term depression or LTP. Thus, in a single preparation, the spatial distribution of NO after induction of hippocampal LTP has been evaluated [Schuppe, et al. 2002]. Main unsolved issues of precise time-point of NO release and selective NOS isoform-inhibitors in neuronal activity still remain to investigate before the combined fluorescent NO-imaging and electrophysiology method becomes an acceptable bioimaging tool. In near future, the researchers will thrive to combine electrophysiology with simultaneous calcium-imaging and NO-imaging to get better answers of the role and interaction of calcium and NO in physiological and pathological neuronal processes with continuous monitoring the calcium and NO by dichroic or multichroic mirrors in synaptic plasticity.

#### **4.7 Alveolar cells**

150 Enzyme Inhibition and Bioapplications

be measured simultaneously [Pittner, et al. 2003]. Other example of monochromator complex is DAQ and DAR-4M AM. These are calcium sensitive non-neurotoxic dyes with unique fluorescent patterns of DAQ (excitation 520 nm; emission 580 nm) and DAR-4M AM (excitation 550 nm; emission 580 nm). These can also combine with the calcium-

A new mechanism was reported in nonparenchymal cells based on Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, was upregulated in vitro and in vivo by nitric oxide (NO) downregulation of BNIP3 gene expression as one mechanism of hepatocyte cell death and liver damage. Authors proposed that inflammatory stresses can lead to the modulation of BNIP3 [Metukuri, et al. 2009].