Doublecortin in the Cerebrospinal Fluid Is a Candidate Biomarker of Neurogenesis after Neonatal Hypoxia Ischemia Brain Injury in Rats

• Published in: Journal of Neurological Surgery Part A: Central European Neurosurgery
• Main Authors: Bregere, C., Fisch, U., Lieb, S., Sailer, M., Chicha, L., Goepfert, F., Kremer, T., Guzman, R.
• Format: Conference Proceeding
• Language: English
• Published: 07.09.2015
• ISSN: 2193-6315; 2193-6323
• DOI: 10.1055/s-0035-1564547

Aims: There is an unmet clinical need to monitor endogenous neurogenesis in vivo in developmental brain disorders. Doublecortin (DCX) is considered as a marker of neurogenesis, because it is highly expressed in migrating neuroblasts. Using a rat model of neonatal hypoxia-ischemia (HI), the aims were to quantify DCX in the cerebrospinal fluid (CSF) of neonates prior to and after an HI brain injury, and to examine whether DCX in the CSF reflects neurogenesis in the brain after HI. Additionally, rat neural stem cells (NSCs) were differentiated in vitro to ascertain that DCX is secreted, and emanates from living, rather than from injured cells. Methods: HI was elicited at postnatal day (P) 7 in Sprague-Dawley rats using the Rice-Vannucci method. Control animals received a sham surgery without HI. CSF was collected before (P5) and after surgery (P10). DCX was quantified using a custom made highly sensitive immunoassay. BrdU was administered from P7 to P9. Animals were sacrificed at P10 and their brains processed for immunohistochemistry. Rat NSCs were isolated from E14.5 embryos, and differentiation was induced by mitogen withdrawal over 15 days. Results: In sham-treated neonates, the concentration of DCX in the CSF (DCX-CSF) decreased by 85% between P5 and P10 ( n  = 11, p  < 0.0001). In contrast, DCX-CSF increased by 123% in HI-injured animals ( n  = 11) during the same time interval. Interaction between postnatal day and treatment was significant (two-way ANOVA p  = 0.0197). In the HI group, a positive correlation between DCX-CSF and stroke severity was observed. DCX immunointensity, and the number of BrdU-positive cells were augmented in the ipsilateral neurogenic niches from HI-injured animals in comparison to sham animals ( p  < 0.01). In vitro, DCX concentration in cell lysate rose significantly over 15 days of differentiation, due to an increasing number of DCX-positive cells. DCX concentration in medium increased concomitantly. Cleaved caspase-3 and DCX co-staining showed limited overlap. Conclusion: The decline in DCX-CSF in sham neonates between P5 and P10 corroborates the reported postnatal downregulation of DCX in the brain. The results after HI suggest that DCX-CSF reflects the neurogenic and proliferative responses in the brain 3 days after the insult. The in vitro data support that doublecortin is released extracellularly from living DCX expressing cells. Overall, DCX in the CSF appears as a valid in vivo biomarker of neurogenesis in the rat model of neonatal HI.