Temporal patterns of circulating cell-free DNA (cfDNA) in a newborn piglet model of perinatal asphyxia

Abstract

Perinatal asphyxia is a severe medical condition resulting from oxygen deficiency (hypoxia) at the time of birth, causing worldwide approximately 680,000 newborn deaths every year. Better prediction of severity of damages including early biomarkers is highly demanded. Elevated levels of circulating cell-free DNA (cfDNA) in blood have been reported for a range of different diseases and conditions, including cancer and prematurity. The objective of this study was to validate methods for assessing cfDNA in blood and cerebrospinal fluid (CSF) and to explore temporal variations in a piglet model of neonatal hypoxia-reoxygenation. Different cfDNA extraction methods in combination with cfDNA detection systems were tested, including a fluorescent assay using SYBR Gold and a qRT-PCR-based technique. Newborn piglets (n = 55) were exposed to hypoxia-reoxygenation, hypoxia-reoxygenation and hypothermia, or were part of the sham-operated control group. Blood was sampled at baseline and at post-intervention, further at 30, 270, and 570 minutes after the end of hypoxia. Applying the fluorescent method, cfDNA concentration in piglets exposed to hypoxia (n = 32) increased from 36.8±27.6 ng/ml prior to hypoxia to a peak level of 61.5±54.9 ng/ml after the intervention and deceased to 32.3±19.1 ng/ml at 570 minutes of reoxygenation, whereas the group of sham-operated control animals (n = 11) revealed a balanced cfDNA profile. Animals exposed to hypoxia and additionally treated with hypothermia (n = 12) expressed a cfDNA concentration of 54.4±16.9 ng/ml at baseline, 39.2±26.9 ng/ml at the end of hypoxia, and of 41.1±34.2 ng/ml at 570 minutes post-intervention. Concentrations of cfDNA in the CSF of piglets exposed to hypoxia revealed at post-intervention higher levels in comparison to the controls. However, these observations were only tendencies and not significant. In a first methodological proof-of-principle study exploring cfDNA using a piglet model of hypoxia-reoxygenation variations in the temporal patterns suggest that cfDNA might be an early indicator for damages caused by perinatal asphyxia.

Quantification of circulating cell-free DNA (cfDNA) in urine using a newborn piglet model of asphyxia

“…(2011) found that variations of absolute DNA values were mainly caused by the use of different isolation methods, with MagNA Pure isolation system (Roche Diagnostics, Mannheim, Germany) yielding highest DNA concentration [31]. We have previously tested different cfDNA isolation kits for blood samples collected from piglets, including the DNeasy Blood & Tissue Kit from Qiagen, NucleoSpin Plasma XS Kit from Macherey-Nagel, and Wizard Genomic DNA Purification Kit from Promega [11]. All the tested methods resulted in very low amounts of cfDNA and we suggested that a direct method for cfDNA isolation should be applied in the future.…”

“…It has been proposed that cfDNA levels in plasma could reflect hypoxic changes in the brain [11], which was based on previous observations of higher concentrations of circulating cfDNA in premature neonates [10]. However, cfDNA is not only present in blood, it has been found in other body fluids, including cerebrospinal fluid (CSF) and urine [11,19,20].…”

“…Higher concentrations of circulating cfDNA have been observed in premature neonates [10] leading to the hypothesis that cfDNA levels in plasma might reflect hypoxic changes in the brain [11]. Akhter et al .…”

“…(2001) used samples from 16 newborn pigs, exposed to varying degrees of hypoxia, revealing that more low-MW (200–2,000 base pairs (bp)) DNA fragments were present in samples with lower ATP (higher degree of hypoxia) [12]. Using a piglet model of neonatal hypoxia-reoxygenation, we have reported temporal variations in cfDNA concentrations in blood in relation to the intervention procedure [11].…”

“…Other sources of cfDNA might be active intra- or extra-cellular release of DNA, leukocyte oxidative burst or extracellular trap formation [15–18]. Remarkably, cfDNA is not only present in blood, it has been found in other body fluids, including cerebrospinal fluid (CSF) and urine [11,19,20]. Measuring cfDNA in urine has several advantages in comparison to the quantification in blood, due to the larger amounts, higher availability, fewer disturbing particles and reduced invasiveness.…”