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Abstract
Research into treatment of stroke and neonatal hypoxic-ischaemic (HI) insult has been beset by high variability in clinical and histopathology outcome despite similar insult severity, necessitating large groups to produce significant results. This is a common problem across species in mice, rats, fetal chick, fetal sheep or newborn piglets and may impair translation of experimental results in clinical trials. Recent studies from our group using magnetic resonance spectroscopy (MRS) demonstrated that several injury biomarkers – Lactate to N-Acetyl Aspartate (Lac/NAA) and Lactate to Creatine (Lac/Cr) ratios in dorsal subcortical white matter (wm) and thalamus (th), as well as whole brain inorganic Phosphate to exchangeable Phosphate Pool (Pi/ePP) show secondary increase within 2–4 h after HI, with apparently almost linear trajectory.
Using data sets from 2 large therapeutic neonatal piglet HI studies – Hypothermia&Xenon (1), and Methyl-Isobutyl-Amiloride (2) – we explored retrospectively whether (A) these early changes can predict MRS and histology (TUNEL, microglial deramification) outcome, and (B) affect statistical quality of the results. Overall, all 5 biomarkers (Pi/ePP, wmLac/Cr, wmLac/NAA, thLac/Cr, thLac/NAA) showed significant correlation with MRS and histology outcome in both studies, but this was particularly moderate for early Pi/ePP and high for thLac/NAA (see figure 1). Moreover, correction for the early thLac/Cr or thLac/NAA using actual/expected ratio revealed a significant difference for the combined Hypothermia&Xenon group versus that of hypothermia alone on most MRS and histology outcome measures.
(A) Tight correlation of early (3.5h post-HI) thalamic Lac/NAA levels and TUNEL+ cell death at 48h in the control (untreated). (B) TUNEL+ cell death–actual values (centre, based on 3.5h thlmLac/NAA and regression line in A), and act/exp ratios (right). Note, compared with all other 3 groups, a significant reduction in act/exp ratios in combined treatment group.
These data suggest that collecting early MRS biomarkers before onset of treatment could reduce variability and improve statistical impact of the final results.