Protection by Inhaled Hydrogen Therapy in a Rat Model of Acute Lung Injury can be Tracked in vivo Using Molecular Imaging

Abstract

Inhaled hydrogen gas (H2) provides protection in rat models of human acute lung injury (ALI). We previously reported that biomarker imaging can detect oxidative stress and endothelial cell death in vivo in a rat model of ALI. Our objective was to evaluate the ability of Tc-hexamethylpropyleneamineoxime (HMPAO) and Tc-duramycin to track the effectiveness of H2 therapy in vivo in the hyperoxia rat model of ALI. Rats were exposed to room air (normoxia), 98% O2 + 2% N2 (hyperoxia) or 98% O2 + 2% H2 (hyperoxia+H2) for up to 60 h. In vivo scintigraphy images were acquired following injection of Tc-HMPAO or Tc-duramycin. For hyperoxia rats, Tc-HMPAO and Tc-duramycin lung uptake increased in a time-dependent manner, reaching a maximum increase of 270% and 150% at 60 h, respectively. These increases were reduced to 120% and 70%, respectively, in hyperoxia+H2 rats. Hyperoxia exposure increased glutathione content in lung homogenate (36%) more than hyperoxia+H2 (21%), consistent with increases measured in Tc-HMPAO lung uptake. In 60-h hyperoxia rats, pleural effusion, which was undetectable in normoxia rats, averaged 9.3 gram/rat, and lung tissue 3-nitrotyrosine expression increased by 790%. Increases were reduced by 69% and 59%, respectively, in 60-h hyperoxia+H2 rats. This study detects and tracks the anti-oxidant and anti-apoptotic properties of H2 therapy in vivo after as early as 24 h of hyperoxia exposure. The results suggest the potential utility of these SPECT biomarkers for in vivo assessment of key cellular pathways in the pathogenesis of ALI and for monitoring responses to therapies.

Figure 1

H&E lung slices from a normoxia rat, and from rats exposed to hyperoxia for 48 hrs and 60 hrs or to hyperoxia+ H₂ for 60 hrs. Top panels are at lower power than lower panels to facilitate assessment of neutrophilic influx and edema. Higher power images were used to assess thickness of the alveolar septum.

Figure 2

Averaged R123 emission signal in mitochondria isolated from lungs of normoxia, 48-hr hyperoxia, and 48-hr hyperoxia+H₂ rats. FWHM = full width, half maximum.

Figure 3

Representative planar images of ^99mTc-HMPAO distribution in a normoxia (left), 48-hr hyperoxia (center) and 48-hr hyperoxia + H₂ (right) rat 20 min following injection. Lung ROI is determined from the ^99mTc-MAA image with the dashed horizontal lower boundary to avoid liver contribution.

Figure 4

Lung uptake of ^99mTc-HMPAO in rats exposed to hyperoxia (filled circles) or hyperoxia+H₂ (open circles) for 24, 48 or 60 hrs. *different from normoxia (time = 0), ^#different from hyperoxia alone, ^&different from hyperoxia pre DEM, ^@different from hyperoxia+H₂ pre DEM, all with p < 0.001. (n) = number of rats: normoxia (7), normoxia + DEM (7), 24-hr hyperoxia (5), 24-hr hyperoxia + DEM (5), 24-hr hyperoxia+H2 (7), 24-hr hyperoxia + H₂ + DEM (7), 48-hr hyperoxia (5), 48-hr hyperoxia +DEM (5), 48-hr hyperoxia +H₂ (8), 48-hr hyperoxia + H₂ + DEM (5), 60-hr hyperoxia (6), 60-hr hyperoxia + DEM (5), 60-hr hyperoxia+H₂ (4), 60-hr hyperoxia + H₂ + DEM (4).

Figure 5

Representative planar images of ^99mTc-duramycin distribution in a normoxia (left), 60-hr hyperoxia (center) and 60-hr hyperoxia+H₂ (right) rat 20 min following injection. Lung ROI is determined from the ^99mTc-MAA image with the dashed horizontal lower boundary to avoid liver contribution.

Figure 6

Lung uptake of ^99mTc-duramycin in rats exposed to hyperoxia (filled circles) or hyperoxia+H₂ (open circles) for 48 or 60 hrs. *different from normoxia (time 0) with p < 0.001, ^#different from hyperoxia alone with p = 0.001 for 48 hrs and p < 0.001 for 60 hrs. (n) = number of rats: normoxia (9), 48-hr hyperoxia (7), 48-hr hyperoxia+H₂ (6), 60-hr hyperoxia (7), 60-hr hyperoxia+H₂ (5).

Figure 7

Relationship between ^99mTc-HMPAO lung uptake (Figure 4) and lung tissue GSH content (as fraction of normoxia, Table 8).