The ex vivo pump systems were able to expel 92.2 ± 6.6% of the target dose. Some animals were able to temporarily dislodge their cannulae. Cannulation was also temporarily halted if animals appeared ill. Nine out of 30 animals displayed clinical signs of ill health during the whole experiment. Ill health was defined as weight loss, dehydration, hunched back, ruffled fur or lethargy. These findings were observed in 8 animals receiving DFO, either alone or in combination, and in 1 cannulated, sham-chelated animal. Upon diagnosis, backpacks with pumps were removed to allow recovery. On average, DFO gerbils missed 3 days and DFO/DFX gerbils about 5 days of DFO chelation therapy for health concerns. Animals in all 3 treatment groups were slightly lighter than control animals, but this difference reached statistical significance only for DFO-treated animals. Control animals weighed 86.9 ± 7.3 g. The body weights of DFX-, DFO/DFX- and DFO-treated animals were 80.5 ± 8.1, 80.6 ± 4.6 and 79.2 ± 5.8 g (p = 0.05), respectively. Total cannulation efficiency was 94.2 ± 4.2% and was not statistically different among the groups.
Organ weight, wet-to-dry weight ratio, dry weight iron concentration and organ iron content are displayed in . Iron loading produces organ hypertrophy in the gerbil [17
]. For reference, normal organ weights in age-matched gerbils are 333 ± 42 mg for the heart and 2.13 ± 0.26 g for the liver; they do not vary significantly with age [19
]. DFX treatment partially normalized liver and cardiac weights relative to controls: 344 ± 31 versus 417 ± 60 mg for the heart and 4.38 ± 0.67 versus 5.94 ± 0.88 g for the liver. The wet-to-dry ratio, cardiac iron concentrations and iron content did not statistically differ among all the groups.
Cardiac and hepatic organ iron contents, dry weight iron concentrations, wet-to-dry weight ratio and organ weights
In the liver, there was no statistically significant change among the treatment groups for the wet-to-dry ratio. DFO treatment alone did not lower hepatic iron concentration or content when compared with the control group. DFX removed more than half of the liver iron, comparable with prior studies. However, combined therapy did not produce an additive effect; in fact, organ weight was higher in the combined therapy animals (5.24 ± 0.6 vs. 4.37 ± 0.67 g for DFX therapy alone).
Cardiac histology was relatively similar among the treatment groups. H&E staining revealed mild to moderate myocardial dense fibers and mild signs of nuclear hypertrophy in the hearts among all the treatment groups (). As reported previously, cardiac iron accumulated mainly in the interstitium (). The only chelator-specific findings were rare misshapen nuclei that were only found in 6 out of 8 animals treated with combined therapy (). Misshapen nuclei were not associated with necrosis. In addition, 1 DFO/DFX-treated animal displayed a focal area of necrosis and inflammation in the left ventricle wall (data not shown).
Fig. 1 Representative histological examination of the heart and liver following 12 weeks of chelation therapy. Specimens were stained with H&E (a – c, e, f) and Perl’s Prussian blue iron stain (d, g, h). Increased appearance of dense (more ...)
Liver histology is highlighted in . DFO-treated and sham-chelated liver histology was similar. In general, there were focal glomular inflammatory infiltrates in areas with masses of hemosiderin (). Prussian blue staining showed increased iron accumulation in hepatocytes, Kupffer cells and infiltrating monocytes (). Heavy iron deposits were observed, in particular around the central veins. DFX and combined treatment caused clear hepatocyte foci devoid of iron (). There were no or little signs of hepatic inflammation. However, cytoplasmic pallor along central veins was prominent in some of the DFX- and DFX/DFO-treated animals (). This phenomenon was also present in 2 control livers.