Neuroprotective effect of dexmedetomidine on hyperoxia-induced toxicity in the neonatal rat brain

Abstract

Dexmedetomidine is a highly selective agonist of α2-receptors with sedative, anxiolytic, analgesic, and anesthetic properties. Neuroprotective effects of dexmedetomidine have been reported in various brain injury models. In the present study, we investigated the effects of dexmedetomidine on neurodegeneration, oxidative stress markers, and inflammation following the induction of hyperoxia in neonatal rats. Six-day-old Wistar rats received different concentrations of dexmedetomidine (1, 5, or 10 µg/kg bodyweight) and were exposed to 80% oxygen for 24 h. Sex-matched littermates kept in room air and injected with normal saline or dexmedetomidine served as controls. Dexmedetomidine pretreatment significantly reduced hyperoxia-induced neurodegeneration in different brain regions of the neonatal rat. In addition, dexmedetomidine restored the reduced/oxidized glutathione ratio and attenuated the levels of malondialdehyde, a marker of lipid peroxidation, after exposure to high oxygen concentration. Moreover, administration of dexmedetomidine induced downregulation of IL-1β on mRNA and protein level in the developing rat brain. Dexmedetomidine provides protections against toxic oxygen induced neonatal brain injury which is likely associated with oxidative stress signaling and inflammatory cytokines. Our results suggest that dexmedetomidine may have a therapeutic potential since oxygen administration to neonates is sometimes inevitable.

Figure 1

Apoptosis caused by hyperoxia is prevented by dexmedetomidine. (a) Representative TUNEL staining images (original magnification ×400) of rat brain frontal cortices (FC), retrosplenial cortices (RSC), hypothalamus (HTH), and thalamus (TH) of P7 control pups in room air without (CON) and with dexmedetomidine administration (DEX1, DEX5, and DEX10, corresponding to the concentrations of 1, 5, and 10 μg/kg) and after 24 h of hyperoxia from P6 to P7 without (HY) and with dexmedetomidine administration (HYDEX1, HYDEX5, and HYDEX10). (b) Quantitation of TUNEL positive cells in the rat brain frontal cortices (FC), retrosplenial cortices (RSC), hypothalamus (HTH), and thalamus (TH) showed that relative to the control (white bars) hyperoxia at 24 h significantly increased these cell counts in cortex and deep grey matter (black bars). These levels were significantly decreased through systemic dexmedetomidine pretreatment (hatched grey bars; DEX 1, 5, and 10 μg/kg). However, dexmedetomidine administration resulted in increased TUNEL positive cells in control rats most profound in TH (grey bars; DEX 1, 5, and 10 μg/kg). Data are expressed relative to the normoxia-exposed control group (white bars, 100%). Bars represent mean + SEM; n = 6 per group; ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001 versus normoxia/control; ^## P < 0.01 and ^### P < 0.001 versus hyperoxia.

Figure 2

Effect of dexmedetomidine on hyperoxia-modified GSH and GSSG levels in the developing brain. (a) Reduced GSH levels were evident in total rat brain extracts 24 h after the initiation of hyperoxia (black bar) when compared to normoxic animals (white bar). These levels were increased through dexmedetomidine (DEX) pretreatment in a concentration dependent manner (hatched grey bars: 1, 5, and 10 μg/kg). (b) Increased levels of oxidized GSSG were obvious in total brain extracts at 24 h of hyperoxia (black bar) when compared with normoxic control animals (white bar). These levels were decreased through pretreatment with DEX (hatched grey bars: 1, 5, and 10 μg/kg). (c) Reduced GSH/GSSG ratio levels were evident in rat brain extracts at 24 h of hyperoxia (black bar) when compared to normoxic control animals (white bar). These levels were upregulated through DEX pretreatment (hatched grey bars: 1, 5, and 10 μg/kg). Application of dexmedetomidine under room air (grey bars) showed no effect on GSH or GSSG levels. Bars represent mean + SEM; n = 6 per group; ^*** P < 0.001 versus normoxia/control; ^### P < 0.001 versus hyperoxia.

Figure 3

Alteration of lipid peroxidation by hyperoxia in the immature brain. Hyperoxia lead to a significant increase of MDA levels after 24 h of oxygen exposure (black bar), whereas a single DEX application of 5 or 10 μg/kg (hatched grey bars) before hyperoxia exposure reduced these levels significantly. Bars represent mean + SEM, n = 6 per group, ^* P < 0.05 and ^*** P < 0.001 versus normoxia/control; ^## P < 0.01 and ^### P < 0.001 versus hyperoxia.

Figure 4

(a) Quantitative analysis of mRNA expression by real-time PCR showed a marked increase of IL-1β mRNA expression in the brain of P6 rat pups that were kept for 24 h under hyperoxia (black bar), whereas dexmedetomidine treatment restores IL-1β upon control level (hatched grey bars) depending on the dexmedetomidine concentration. Application of dexmedetomidine under room air (grey bars) showed no significant regulation on IL-1β mRNA expression. (b) The analysis of IL-1β protein expression by western blot showed a similar expression pattern. The protein expression of IL-1β is significantly increased after 24 h of hyperoxia and a single application of 5 or 10 μg/kg dexmedetomidine could restore the IL-1β protein expression almost up to control level. The densitometric data represent the ratio of the pixel intensity of the IL-1β band to the corresponding β-actin band. Blots are representative of a series of three blots. Data are normalized to levels of rat pups exposed to normoxia (CON = 100%, white bars). Bars represent mean + SEM; n = 6 per group; ^*** P < 0.001 versus normoxia/control; ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001 versus hyperoxia.