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

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

indicating that relaxivity is not magnetic field dependent over this range.

r1 L.mmol-1.sec-1

r2 L.mmol-1.sec-1

 spin-trapped NO is stable in intracellular tissue environment; NO-LPS contrast enhancement properties are MRI visible; NO-LPS complex is stable in tissues and organs; and

*In vivo* Imaging Approaches of Nitric Oxide with Multimodal Imaging 127

The routine method of LPS injected animals after 6 hours of (MGD)2-Fe(II) injection, usually serve as experimental model of subsequent MRI detection and visualization of NO generated in animals. It was confirmed by using a specific NOS inhibitor *N*-monomethyl Larginine (L-NMMA) [Fujii, et al. 2007]. The observations indicated that the suppressed NO levels were proportional to NO sensitive LPS concentration independent of (MGD)2-Fe(II)- NO contrast agent [Fujii, et al. 2007]. Next issue of feasibility and stability of (MGD)2-Fe(II)- NO imaging contrast agent was major breakthrough reported in model systems to generate EPR and MRI signals. It is established that the NO complex (MGD)2-Fe(II)-NO is very stable in model aqueous media. The NO complex (MGD)2-Fe(II)-NO complex, if injected, is stable in tissues and organs as confirmed by L-band EPR measurements as reported elsewhere [Lai, et al. 1994, Mulsch, et al. 1999]. These authors confirmed the assignment as (MGD)2- Fe(II)-NO from the hyperfine coupling J constant of this signal (see Figure 5). However, LPS complex accumulates in liver, brain, heart, kidney [Yoshimura, et al. 1996]. NOS inhibitor, *N*-monomethyl L-arginine (L-NMMA, 2 mM) confirmed that the NO complex is not bioreduced or biodegraded *in vivo* based on the EPR spectrum of (MGD)2-Fe(II)-NO complex. The stability of (MGD)2-Fe(II)-NO complex was at least 12 hours. In other report, the reduction/ decomposition of the NO complex occurred in the presence of 1 mM ascorbic acid or glutathione inhibitor with a half-life of 40 and 48 min, respectively [Perrier, et al. 2009]. All these evidences indicate the NO complex as stable, non-biodegradable or decomposed form of (MGD)2-Fe(II)-NO complex as strong proton relaxation enhancer with paramagnetic properties [Perrier, et al. 2009, Fujii, et al. 2007]. All these evidences also indicated the NO complex as stable, non-biodegradable or decomposed form of (MGD)2- Fe(II)-NO complex as strong proton relaxation enhancer with paramagnetic properties.

simultaneous visualization and mapping of NO free radicals are possible by MRI.

superoxide anion. Peroxynitrites promote apoptosis through the indirect activation of caspases [Kuppusamy, et al. 1994, Hortelano, et al.2005]. However, all these approaches are still invasive to evaluate apoptosis.

Overproduction of nitric oxide and imaging the NO accumulation due to neurotoxicity was reported in neurological disorders or neurodegeneration. The reason of neurotoxicity was reported due to NO reactive oxidative properties in tissue [Caramia, et al. 1998]. This approach was reported not to solve the purpose of *in vivo* functional monitoring but NO properties indicated the state of tissue and follow-up of dynamic status of neurodegenerative factors in brain [Caramia, et al. 1998]. In other application of NO bioimaging to detect apoptosis, T2 weighted maps from control vs GSNO treated and z.VAD treated animals exhibited hyperintense areas perhaps due to toxicity of NO on the T2 maps while z-VAD treated animals showed small lesion areas due to reduced NO toxicity. The response of z-VAD injection was presumed as reduced toxicity due to caspases and NOdependent apoptosis. These authors explained that GSNO increased T2 intensity 25% while z-VAD reduced this MRI signal [Komarov, et al. 1995]. These classic reports indicated that T2 hyperintensities on MRI positively offer the possibility of in vivo evaluation of cell death at different locations in whole tissues undergoing apoptosis. The apoptosis is also a major mechanism in brain neurodegeneration and post-myocardial infarction heart [Caramia, et al. 1998]. However, this approach seems as potential tool of functional imaging in near future to monitor the therapeutic intervention of new drugs to reduce neurodegeneration[ Kuppusamy, et al. 1994, Hortelano, et al.2005, Foster, et al. 1998]. Currently, non-invasive NO sensitive techniques are big hope as potential and remarkable tools to detect apoptosis *in vivo*.

Initial studies on NO with iron-dithiocarbamate complexes had succeeded in direct detection of NO in mice by whole body electron paramagnetic resonance spectroscopy (EPR) at the L-band [Fujii, et al. 1997, Komarov, et al. 1995] with new possibilities by Magnetic Resonance Spectroscopy [Reif, et al. 2009, Li, et al. 2009]. Several authors demonstrated the feasibility of EPR imaging in visualizing free radical distributions *in vivo* at low resolution where the intrinsic line width of the radical is large, such as the spintrapped NO [Yoshimura, et al. 1996, Kubrina, et al. 1992, Foster, et al. 1998]. This drawback of low resolution caught attention for feasible MRI contrast agents, such as stable (*N*-methyl-D-glucamine)2-Fe(II)-NO complex to generate better resolution. The complex has a much longer *in vivo* half-life than most (stable) nitric oxide derived compounds. Recently Nmethyl glucamine iron complexes have shown greater affinity with NO to make (MGD)2- Fe(II)-NO complex useful for in vivo NO measurements by EPR [Caramia, et al. 1998]. In recent years, the art of MRI combined with NO spin trapping mechanism was evaluated as feasible method of mapping the distribution of NO spin-trap complex in animals with possibility in clinical use. In following section, our design of ultrafast MRI protocol is described using NO/NOS sensitive biosensor complexes for use at 21-Tesla MRI microimager.

#### **2.3 Approach of stable NO sensitive lipopolysaccharides (LPS) as feasible imaging complexes**

The LPS serves as encaged bag holding contrast agent. The LPS based NO imaging approach has following presumptions:

superoxide anion. Peroxynitrites promote apoptosis through the indirect activation of caspases [Kuppusamy, et al. 1994, Hortelano, et al.2005]. However, all these approaches are

Overproduction of nitric oxide and imaging the NO accumulation due to neurotoxicity was reported in neurological disorders or neurodegeneration. The reason of neurotoxicity was reported due to NO reactive oxidative properties in tissue [Caramia, et al. 1998]. This approach was reported not to solve the purpose of *in vivo* functional monitoring but NO properties indicated the state of tissue and follow-up of dynamic status of neurodegenerative factors in brain [Caramia, et al. 1998]. In other application of NO bioimaging to detect apoptosis, T2 weighted maps from control vs GSNO treated and z.VAD treated animals exhibited hyperintense areas perhaps due to toxicity of NO on the T2 maps while z-VAD treated animals showed small lesion areas due to reduced NO toxicity. The response of z-VAD injection was presumed as reduced toxicity due to caspases and NOdependent apoptosis. These authors explained that GSNO increased T2 intensity 25% while z-VAD reduced this MRI signal [Komarov, et al. 1995]. These classic reports indicated that T2 hyperintensities on MRI positively offer the possibility of in vivo evaluation of cell death at different locations in whole tissues undergoing apoptosis. The apoptosis is also a major mechanism in brain neurodegeneration and post-myocardial infarction heart [Caramia, et al. 1998]. However, this approach seems as potential tool of functional imaging in near future to monitor the therapeutic intervention of new drugs to reduce neurodegeneration[ Kuppusamy, et al. 1994, Hortelano, et al.2005, Foster, et al. 1998]. Currently, non-invasive NO sensitive techniques are big hope as potential and remarkable tools to detect apoptosis

Initial studies on NO with iron-dithiocarbamate complexes had succeeded in direct detection of NO in mice by whole body electron paramagnetic resonance spectroscopy (EPR) at the L-band [Fujii, et al. 1997, Komarov, et al. 1995] with new possibilities by Magnetic Resonance Spectroscopy [Reif, et al. 2009, Li, et al. 2009]. Several authors demonstrated the feasibility of EPR imaging in visualizing free radical distributions *in vivo* at low resolution where the intrinsic line width of the radical is large, such as the spintrapped NO [Yoshimura, et al. 1996, Kubrina, et al. 1992, Foster, et al. 1998]. This drawback of low resolution caught attention for feasible MRI contrast agents, such as stable (*N*-methyl-D-glucamine)2-Fe(II)-NO complex to generate better resolution. The complex has a much longer *in vivo* half-life than most (stable) nitric oxide derived compounds. Recently Nmethyl glucamine iron complexes have shown greater affinity with NO to make (MGD)2- Fe(II)-NO complex useful for in vivo NO measurements by EPR [Caramia, et al. 1998]. In recent years, the art of MRI combined with NO spin trapping mechanism was evaluated as feasible method of mapping the distribution of NO spin-trap complex in animals with possibility in clinical use. In following section, our design of ultrafast MRI protocol is described using NO/NOS sensitive biosensor complexes for use at 21-Tesla MRI

**2.3 Approach of stable NO sensitive lipopolysaccharides (LPS) as feasible imaging** 

The LPS serves as encaged bag holding contrast agent. The LPS based NO imaging

still invasive to evaluate apoptosis.

*in vivo*.

microimager.

**complexes** 

approach has following presumptions:


The routine method of LPS injected animals after 6 hours of (MGD)2-Fe(II) injection, usually serve as experimental model of subsequent MRI detection and visualization of NO generated in animals. It was confirmed by using a specific NOS inhibitor *N*-monomethyl Larginine (L-NMMA) [Fujii, et al. 2007]. The observations indicated that the suppressed NO levels were proportional to NO sensitive LPS concentration independent of (MGD)2-Fe(II)- NO contrast agent [Fujii, et al. 2007]. Next issue of feasibility and stability of (MGD)2-Fe(II)- NO imaging contrast agent was major breakthrough reported in model systems to generate EPR and MRI signals. It is established that the NO complex (MGD)2-Fe(II)-NO is very stable in model aqueous media. The NO complex (MGD)2-Fe(II)-NO complex, if injected, is stable in tissues and organs as confirmed by L-band EPR measurements as reported elsewhere [Lai, et al. 1994, Mulsch, et al. 1999]. These authors confirmed the assignment as (MGD)2- Fe(II)-NO from the hyperfine coupling J constant of this signal (see Figure 5). However, LPS complex accumulates in liver, brain, heart, kidney [Yoshimura, et al. 1996]. NOS inhibitor, *N*-monomethyl L-arginine (L-NMMA, 2 mM) confirmed that the NO complex is not bioreduced or biodegraded *in vivo* based on the EPR spectrum of (MGD)2-Fe(II)-NO complex. The stability of (MGD)2-Fe(II)-NO complex was at least 12 hours. In other report, the reduction/ decomposition of the NO complex occurred in the presence of 1 mM ascorbic acid or glutathione inhibitor with a half-life of 40 and 48 min, respectively [Perrier, et al. 2009]. All these evidences indicate the NO complex as stable, non-biodegradable or decomposed form of (MGD)2-Fe(II)-NO complex as strong proton relaxation enhancer with paramagnetic properties [Perrier, et al. 2009, Fujii, et al. 2007]. All these evidences also indicated the NO complex as stable, non-biodegradable or decomposed form of (MGD)2- Fe(II)-NO complex as strong proton relaxation enhancer with paramagnetic properties.

