Neuroprotective effects of dexmedetomidine against hyperoxia-induced injury in the developing rat brain

Abstract

Dexmedetomidine (DEX) is a highly selective agonist of α2-receptors with sedative, anxiolytic, and analgesic properties. Neuroprotective effects of dexmedetomidine have been reported in various brain injury models. In the present study, we investigated the effects of dexmedetomidine on hippocampal neurogenesis, specifically the proliferation capacity and maturation of neurons and neuronal plasticity following the induction of hyperoxia in neonatal rats. Six-day old sex-matched Wistar rats were exposed to 80% oxygen or room air for 24 h and treated with 1, 5 or 10 μg/kg of dexmedetomidine or normal saline. A single pretreatment with DEX attenuated the hyperoxia-induced injury in terms of neurogenesis and plasticity. In detail, both the proliferation capacity (PCNA+ cells) as well as the expression of neuronal markers (Nestin+, PSA-NCAM+, NeuN+ cells) and transcription factors (SOX2, Tbr1/2, Prox1) were significantly reduced under hyperoxia compared to control. Furthermore, regulators of neuronal plasticity (Nrp1, Nrg1, Syp, and Sema3a/f) were also drastically decreased. A single administration of dexmedetomidine prior to oxygen exposure resulted in a significant up-regulation of expression-profiles compared to hyperoxia. Our results suggest that dexmedetomidine may have neuroprotective effects in an acute hyperoxic model of the neonatal rat.

Fig 1. Paraffin sections of the hippocampus at postnatal day (P)7 of Wistar rats stained with A/B) proliferating cell nuclear antigen (PCNA)/DAPI; double immunofluorescence staining with C/D) Nestin/PCNA/DAPI; E/F) polysialylated neuronal cell adhesion molecule (PSA-NCAM)/PCNA/DAPI; G/H) neuronal nuclei (NeuN)/PCNA/DAPI.

Hyperoxia in neonatal rats decreased the proliferation positive cells (PCNA+, green, A/B) and the expression of neuronal marker for neuronal progenitor cells (Nestin+, red, C/D), immature neurons (PSA-NCAM+, red, E/F), and mature postmitotic neurons (NeuN+, red, G/H). Application of DEX1 and/or DEX5 under hyperoxic exposure resulted in improved expression of neuronal markers and increase of proliferation in granular cell layer and polymorphic layer of DG. DEX10 led to a reduction of cell counts of NeuN in hyperoxic animals. Under normoxic conditions DEX1 and/or DEX5 upregulated PCNA and the differentiation marker and DEX10 showed negative effects on PNCA and NeuN expression. All images were taken at identical magnification (original magnification 200 x).

Fig 2. Quantitation of A) PCNA+, B) Nestin+, C) PSA-NCAM+, and D) NeuN+ cell counts in sum of the granular cell layer and polymorphic layer with DEX (1, 5, and 10 μg/kg) under hyperoxia (hatched grey bars), hyperoxia alone (black bars), DEX under normoxic conditions (plain grey bars), and in comparison to normoxia control group (100%, white bars).

Data are expressed relative to the normoxia-exposed control group as mean ± SEM of n = 5. The 100% values are for PCNA 258.9, for Nestin 354.1, for PSA-NCAM 209.3, and for NeuN 333.3. * p<0.05, ** p<0.01, and *** p<0.001 versus control; ^### p<0.001 versus hyperoxia (t-test, Bonferroni post hoc test after one-way ANOVA).

Fig 3. Expression of mediators of transcriptional network is decreased in neonatal rats after hyperoxic injury and upregulated with DEX.

The relative mRNA expressions of transcription factors were measured in rat brain homogenates with DEX (1, 5, and 10 μg/kg) under hyperoxia (hatched grey bars), hyperoxia alone (black bars), DEX under normoxic conditions (plain grey bars), and in comparison to normoxia control group (100%, white bars) by quantitative realtime PCR. A) There are no changes under normoxia/hyperoxia with or without DEX for Pax6. B) Note the significant reduction of SOX2 under hyperoxia and the increase with a single dose of DEX. Under normoxic conditions with DEX1 and DEX10 the mRNA expression of SOX2 was increased significantly. C) Tbr2 mRNA expression was reduced under hyperoxia and upregulated at DEX5. D) Hyperoxia has no influence on the mRNA expression of Tbr1, but DEX5 and DEX10 triggered the expression above the normoxia level. The same effect was detected for DEX10 under normoxia. E) There was a significant decrease for Prox1 mRNA expression under hyperoxia alone and with DEX1 and DEX5, but also a significant up-regulation with DEX10. Data shown as mean ± SEM, n = 5 per group. * p<0.05, ** p<0.01, and *** p<0.001 versus control; ^## p<0.01 and ^### p<0.001 versus hyperoxia (t-test, Bonferroni post hoc test after one-way ANOVA).

Fig 4. High oxygen concentration resulted in drastically reduced mRNA und protein expression of factors for neurodevelopment and plasticity, and DEX administration reversed these effects.

The relative protein and mRNA expression of plasticity factors (Nrp1/NRP1, Nrg1/NRG1, Syp/SYP, Sema3a/SEMA3A, and Sema3f/SEMA3F) were measured in brain homogenates with DEX application (1, 5, and 10 μg/kg) under hyperoxia (hatched grey bars), hyperoxia alone (black bars), DEX under normoxic conditions (plain grey bars), and in comparison to the normoxia control group (100%, white bars) by quantitative realtime PCR and Western blot. We detected in all plasticity factors a significant decrease under hyperoxic conditions for A-E) mRNA expression and F-J) protein expression. The application of DEX5 and DEX10 under hyperoxia resulted in a significant increase of expression. No changes were measured under normoxia with DEX. Data shown as mean ± SEM, n = 5 per group. ** p<0.01, and *** p<0.001 versus control; ^# p<0.05, ^## p<0.01 and ^### p<0.001 versus hyperoxia (t-test, Bonferroni post hoc test after one-way ANOVA).

Fig 5. Generation of new hippocampal neurons in the developing brain and modulation with hyperoxia and dexmedetomidine.

A) Radialglia-like stem cells undergo different stages of processing with proliferation and generation of neural progenitors that can further differentiate into mitotic and postmitotic neurons and finally into mature neurons. These well-orchestrated processes are modulated through different intrinsic factors and it also characterized something like transcription factors and neuronal markers. B) The results of our experimental animal study demonstrated impressively the downregulation of neurogenesis- and plasticity-related factors by acute hyperoxia (red square) of the six-day old rat pups compared to normoxia exposure (green rhombus). The α2 agonist dexmedetomidine at concentrations of 5 μg/kg (light blue triangle) and 10 μg/kg (blue triangle) was significantly protective against oxidative stress in the process of neurogenesis and neuroplasticity. GCL, granule cell layer; ML, molecular layer; NeuN, neuronal nuclei; Nrg1, neuregulin 1; Nrp1, neuropilin 1; Pax6, paired box 6; PCNA, proliferating cell nuclear antigen; PL, polymorphic layer; Prox1, prospero homeobox 1; PSA-NCAM, polysialylated neuronal cell adhesion molecule; Sema3a/f, semaphorin3a/f; SOX2, sex determining region Y-box 2; SGZ, subgranular zone; Syp, synaptophysin; Tbr1/2, T-box brain gene 1/2.