Resuscitation of newborn piglets. short-term influence of FiO2 on matrix metalloproteinases, caspase-3 and BDNF

Abstract

Background: Perinatal hypoxia-ischemia is a major cause of mortality and cerebral morbidity, and using oxygen during newborn resuscitation may further harm the brain. The aim was to examine how supplementary oxygen used for newborn resuscitation would influence early brain tissue injury, cell death and repair processes and the regulation of genes related to apoptosis, neurodegeneration and neuroprotection.

Methods and findings: Anesthetized newborn piglets were subjected to global hypoxia and then randomly assigned to resuscitation with 21%, 40% or 100% O(2) for 30 min and followed for 9 h. An additional group received 100% O(2) for 30 min without preceding hypoxia. The left hemisphere was used for histopathology and immunohistochemistry and the right hemisphere was used for in situ zymography in the corpus striatum; gene expression and the activity of various relevant biofactors were measured in the frontal cortex. There was an increase in the net matrix metalloproteinase gelatinolytic activity in the corpus striatum from piglets resuscitated with 100% oxygen vs. 21%. Hematoxylin-eosin (HE) staining revealed no significant changes. Nine hours after oxygen-assisted resuscitation, caspase-3 expression and activity was increased by 30-40% in the 100% O(2) group (n = 9/10) vs. the 21% O(2) group (n = 10; p<0.04), whereas brain-derived neurotrophic factor (BDNF) activity was decreased by 65% p<0.03.

Conclusions: The use of 100% oxygen for resuscitation resulted in increased potentially harmful proteolytic activities and attenuated BDNF activity when compared with 21%. Although there were no significant changes in short term cell loss, hyperoxia seems to cause an early imbalance between neuroprotective and neurotoxic mechanisms that might compromise the final pathological outcome.

Figure 1. Brain histopathology.

Typical morphological changes after hypoxia and reoxygenation. HE-stained sections from the corpus striatum with vacuolated neuropil (black arrow), shrunken neurons with pyknotic nuclei (white arrow), and eosinophilic neurons (arrow head) from one representative animal in each group together with HE stained sections from the control- and hyperoxia group. Obj. ×20.

Figure 2. In situ zymography in the corpus striatum.

Net gelatinolytic activity increases in the striatum after hypoxia-resuscitation. Fluorescence photomicrographs of striatum sections showing in situ zymography in sham operated (Ctl) and hyperoxia (Hyp) controls and after reoxygenation with 21%, 40% or 100% O₂. Overall, fluorescence signal representing proteolytic activity (green) increases after hypoxia-resuscitation in the entire tissue, but the most prominent changes occur in discrete neuronal populations (arrows) in a dose-response manner. Hoechst stain was used as a nuclear marker (blue). Scale bar: 150 µm. The graph represents the quantification of net gelatinolytic activity (in arbitrary units (AU) of fluorescence) for 21% (n = 8) 50.64 (10.1), 40% (n = 8) 55.29 (5.4), 100% (n = 9) 59.51 (11.1), hyperoxia (n = 6) 35.42 (8.0) and controls (n = 6) 35.67 (6.1). There was a significant increase in net gelatinolytic activity in the corpus striatum in all groups exposed to hypoxia-reoxygenation vs the control group (p = 0.024, p = 0.002 and p<0.001 for the 21%, 40% and 100% group). The hyperoxia-group was similar to the control group. Using post hoc multiple comparisons between group means (Fisher LSD), we found a significant increase in the 100% oxygen group compared to the 21% oxygen group (p = 0.043). Values are expressed as a mean (±SD), *p<0.05, **p<0.01.

Figure 3. In situ zymography.

Net gelatinase activity at the nuclear level. High power magnification of fluorescence photomicrographs of the striatum sections showing in situ zymography (green) and nuclear marker Hoechst (blue) in sham operated (Ctl) and after reoxygenation with 40% or 100% O₂. Note that, the number of cells showing intense gelatinolysis in the nucleus (arrows) augments with the concentration of oxygen. Scale bar 25 µm.

Figure 4. MMP-9 mRNA expression in the cortex.

Relative mRNA MMP-9 expression was significantly increased in the 21% oxygen group vs. all the others: 21% (n = 10) 23.1 (19.3), 40% (n = 12) 7.8 (15.1), 100% (n = 10) 4.3 (4.2), hyperoxia (n = 11)1.8 (1.6), control (n = 6) 3.9 (4) with p = 0.029, 0.007, 0.001 and 0.017, respectively.

Figure 5. BDNF and caspase-3.

A: For BDNF, the relative mRNA expression for each group was: 21% (n = 10) 914.6 (909), 40% (n = 10) 585.1 (642.8), 100% (n = 10) 466.5 (412.3) p = 0.15 for 21% vs. 100% due to great inter-animal variability.The hyperoxia group (n = 10) was equal to the control group (n = 6); 391.7 (374) vs. 406.2 (320). B: Immunohistochemistry (ELISA). BDNF (pg/mg protein) was 21% (n = 10) 187.5 (149.1), 40% (n = 12) 105.2 (93.3), 100% (n = 10) 66.4 (45.2). * p = 0.028 for 21% vs. 100%. The hyperoxia group (n = 11) was equal to the control group (n = 6); 85.3 (36.0) vs. 94.4 (53.5). C: For caspase-3, the relative mRNA expression for each group was: 21% (n = 10) (26.0 (8.4), 40% (n = 12) 34.0 (14.5), 100% (n = 9) 33.7 (6.5). *p = 0.037 for 21% vs. 100%. The hyperoxia group (n = 11) was equal to the control group (n = 6); 27.4 (8.7) vs. 31.2 (11.7). D: Immunohistochemistry (ELISA). Caspase-3 (pg/mg protein) was 21% (n = 10)136.1 (80.6), 40% (n = 12)195.1 (94.6), 100% (n = 10)188.9 (64.8). * p = 0.037 for 21% vs. 40% and *p = 0.039 for 21% vs. 100%. The hyperoxia group (n = 11) was equal to the control group (n = 6); 258.7 (97.5) vs. 279.6 (58.0). Caspase-3 expression levels correlated negatively with BDNF expression levels (r = −0.49, p = 0.024). Values are expressed as mean (±SD), *p<0.05.