Dexmedetomidine attenuates the propofol-induced long-term neurotoxicity in the developing brain of rats by enhancing the PI3K/Akt signaling pathway

Figures & data

Figure 1 The experimental flowchart.

Notes: (A) The experimental grouping method. All pups were assigned to 13 groups by a random number table (n=30 per group). P group: i.p. injected with 50 mg/kg propofol, and 40–60 min later, when the righting reflex was recovered, the procedure was repeated one more time; PD₂₅, PD₅₀ and PD₇₅ groups: intraperitoneal injection with 25, 50 and 75 μg/kg Dex, and 30 min later, intervention with propofol; LPD group: intracerebroventricular injection with 25 μg/5 μL LY294002, and 30 min later, intervention with Dex and propofol; TPD group: intraperitoneal injection with 1 mg/kg TDZD-8, and 30 min later, intervention with Dex and propofol. DS₁ and DS₂ groups: intracerebroventricular or intraperitoneal injection with DMSO; F group: intraperitoneal injection with intralipid; L group: intracerebroventricular injection with LY294002; T group: intraperitoneal injection with TDZD-8; Dex group: intraperitoneal injection with Dex. (B) The experimental steps. The pups were intervened at postnatal 7 days. An hour post-recovery from the last injection, five pups in each group were subjected to arterial blood gas analysis, and other rats were returned to their mothers until weaning at 4 weeks of age. Morris water maze test, TUNEL, immunohistochemistry, Western blot and TEM assays were conducted at 9 weeks of age.
Abbreviations: Dex, dexmedetomidine; DMSO, dimethyl sulfoxide; i.c., intracerebroventricularly; i.p., intraperitoneally; TEM, transmission electron microscopy; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 2 Injection of young rats with Dex and/or propofol did not affect their body weights in adult age.

Notes: Young rats were injected with propofol or Dex either alone or in combination with another drug, and the body weights of different groups of rats were monitored longitudinally. Data are expressed as the mean±SEM of each group at different time points (n=25 per group). P group: i.p. injected with 50 mg/kg propofol, and 40–60 min later, when the righting reflex was recovered, the procedure was repeated one more time; PD₂₅, PD₅₀ and PD₇₅ groups: intraperitoneal injection with 25, 50 and 75 μg/kg Dex, and 30 min later, intervention with propofol; LPD group: intracerebroventricular injection with 25 μg/5 μL LY294002, and 30 min later, intervention with Dex and propofol; TPD group: intraperitoneal injection with 1 mg/kg TDZD-8, and 30 min later, intervention with Dex and propofol. DS₁ and DS₂ groups: intracerebroventricular or intraperitoneal injection with DMSO; F group: intraperitoneal injection with intralipid; L group: intracerebroventricular injection with LY294002; T group: intraperitoneal injection with TDZD-8; Dex group: intraperitoneal injection with Dex.
Abbreviations: Dex, dexmedetomidine; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.

Figure 3 Pretreatment of rats with Dex at young age improved the propofol-induced impairment in spatial learning and memory ability in adult age.

Notes: P group: i.p. injected with 50 mg/kg propofol, and 40–60 min later, when the righting reflex was recovered, the procedure was repeated one more time; PD₂₅, PD₅₀ and PD₇₅ groups: intraperitoneal injection with 25, 50 and 75 μg/kg Dex, and 30 min later, intervention with propofol; LPD group: intracerebroventricular injection with 25 μg/5 μL LY294002, and 30 min later, intervention with Dex and propofol; TPD group: intraperitoneal injection with 1 mg/kg TDZD-8, and 30 min later, intervention with Dex and propofol. DS₁ and DS₂ groups: intracerebroventricular or intraperitoneal injection with DMSO; F group: intraperitoneal injection with intralipid; L group: intracerebroventricular injection with LY294002; T group: intraperitoneal injection with TDZD-8; Dex group: intraperitoneal injection with Dex. Young rats were pretreated with Dex or another indicated compound and injected with propofol. At 9 weeks of age, the different groups of rats were trained for 4 days and tested by MWM. The escape latency time and swimming speed of individual rats were recorded. Data are expressed as the mean±SEM of each group (n=8) from three separate experiments. (A) The escape latency time. (B) The swimming speed. (C) The escape latency time on the first training day. (D) The escape latency time on the testing day. ^*p<0.05 versus the NS group; ^#p<0.05 versus the P group; ^p<0.05 versus the PD₇₅ group; double symbols mean p<0.01.
Abbreviations: Dex, dexmedetomidine; DMSO, dimethyl sulfoxide; MWM, Morris water maze; SEM, standard error of the mean.

Table 1 Injection with Dex and/or propofol did not affect arterial blood gas

Group	pH	PaO2 (mmHg)	PaCO2 (mmHg)	HCO3^− (mmol/L)
NS	7.30±0.02	92.6±5.6	44.3±4.5	23.7±2.4
P	7.28±0.02	89.2±8.4	50.4±5.1	22.5±2.4
PD25	7.29±0.03	89.0±9.4	48.7±5.7	23.6±2.6
PD50	7.28±0.02	91.8±7.4	50.9±6.3	23.1±3.4
PD75	7.28±0.03	89.2±8.2	46.6±5.3	23.1±3.4
LPD	7.30±0.02	89.6±7.8	49.4±4.5	22.8±2.7
TPD	7.29±0.03	89.0±7.5	48.9±2.9	22.8±2.8
F	7.30±0.02	91.4±8.0	44.9±4.7	23.8±2.9
DS1	7.30±0.02	91.4±8.0	45.1±4.5	24.6±3.1
DS2	7.29±0.01	94.0±5.7	44.4±4.8	23.9±3.0
Dex	7.30±0.02	92.4±7.8	43.3±5.6	23.5±2.9
L	7.30±0.02	89.60±6.8	46.6±5.8	22.9±2.6
T	7.30±0.02	90.6±7.1	45.9±4.6	22.8±2.5

Notes: NS group: saline; P group: propofol; PD₂₅, PD₅₀ and PD₇₅ groups: pretreatment with Dex, injection with propofol; LPD group: pretreatment with LY294002 and Dex, injection with propofol; TPD group: pretreatment with TDZD-8 and Dex, injection with propofol; F group: intralipid; DS₁ group: intracerebroventricular injection with DMSO; DS₂ group: intraperitoneal injection with DMSO; Dex group: Dex; L group: intracerebroventricular injection with LY294002; T group: TDZD-8. Results are presented as the mean±SEM (n=5 per group).

Abbreviations: Dex, dexmedetomidine; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.

Figure 4 Pretreatment of young rats with Dex decreased the propofol-induced neuroapoptosis in the hippocampus in adult age.

Notes: P group: i.p. injected with 50 mg/kg propofol, and 40–60 min later, when the righting reflex was recovered, the procedure was repeated one more time; PD₂₅, PD₅₀ and PD₇₅ groups: intraperitoneal injection with 25, 50 and 75 μg/kg Dex, and 30 min later, intervention with propofol; LPD group: intracerebroventricular injection with 25 μg/5 μL LY294002, and 30 min later, intervention with Dex and propofol; TPD group: intraperitoneal injection with 1 mg/kg TDZD-8, and 30 min later, intervention with Dex and propofol. DS₁ and DS₂ groups: intracerebroventricular or intraperitoneal injection with DMSO; F group: intraperitoneal injection with intralipid; L group: intracerebroventricular injection with LY294002; T group: intraperitoneal injection with TDZD-8; Dex group: intraperitoneal injection with Dex. The frequency of apoptotic cells in the hippocampus of individual rats was determined by TUNEL assay using FITC for apoptotic cells and DAPI for nucleus staining. Data are representative images (magnification ×200) or are expressed as the mean±SEM of each group (n=5 per group) from three separate experiments. ^*p<0.05 versus NS group; ^#p<0.05 versus P group; ^p<0.05 versus PD₇₅ group; double symbols mean p<0.01. The red arrow on each merged picture pointed to the neuroapoptosis, and the red box on the lower right corner was its larger image.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Dex, dexmedetomidine; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; SEM, standard error of the mean; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Figure 5 Pretreatment of young rats with Dex attenuated the propofol-induced long-term neurotoxicity and increased the levels of PSD₉₅ expression in the hippocampus in adult age.

Notes: P group: i.p. injected with 50 mg/kg propofol, and 40–60 min later, when the righting reflex was recovered, the procedure was repeated one more time; PD₂₅, PD₅₀ and PD₇₅ groups: intraperitoneal injection with 25, 50 and 75 μg/kg Dex, and 30 min later, intervention with propofol; LPD group: intracerebroventricular injection with 25 μg/5 μL LY294002, and 30 min later, intervention with Dex and propofol; TPD group: intraperitoneal injection with 1 mg/kg TDZD-8, and 30 min later, intervention with Dex and propofol. DS₁ and DS₂ groups: intracerebroventricular or intraperitoneal injection with DMSO; F group: intraperitoneal injection with intralipid; L group: intracerebroventricular injection with LY294002; T group: intraperitoneal injection with TDZD-8; Dex group: intraperitoneal injection with Dex. The levels of PSD₉₅ expression in the hippocampus of individual rats were examined by immunohistochemistry using anti-PSD₉₅, HRP-conjugated secondary antibodies and DAB. Data are representative images (magnification ×200) or are expressed as the mean±SEM of each group (n=5 per group) from three separate experiments. ^*p<0.05 versus NS group; ^#p<0.05 versus P group; ^p<0.05 versus PD₇₅ group; double symbols mean p<0.01.
Abbreviations: DAB, 3,3′-diaminobenzidine tetrahydrochloride; Dex, dexmedetomidine; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; SEM, standard error of the mean.

Figure 6 Western blot analysis of the relative levels of PSD₉₅ expression in the hippocampus of rats.

Notes: P group: i.p. injected with 50 mg/kg propofol, and 40–60 min later, when the righting reflex was recovered, the procedure was repeated one more time; PD₂₅, PD₅₀ and PD₇₅ groups: intraperitoneal injection with 25, 50 and 75 μg/kg Dex, and 30 min later, intervention with propofol; LPD group: intracerebroventricular injection with 25 μg/5 μL LY294002, and 30 min later, intervention with Dex and propofol; TPD group: intraperitoneal injection with 1 mg/kg TDZD-8, and 30 min later, intervention with Dex and propofol. DS₁ and DS₂ groups: intracerebroventricular or intraperitoneal injection with DMSO; F group: intraperitoneal injection with intralipid; L group: intracerebroventricular injection with LY294002; T group: intraperitoneal injection with TDZD-8; Dex group: intraperitoneal injection with Dex. (A) The relative levels of PSD₉₅ expression in the hippocampus of individual rats were determined by Western blot assay. (B) Data are representative images or are expressed as the mean±SEM of each group (n=5 per group) from three separate experiments. ^*p<0.05 versus NS group; ^#p<0.05 versus P group; ^p<0.05 versus PD₇₅ group; double symbols mean p<0.01.
Abbreviations: DMSO, dimethyl sulfoxide; sSEM, standard error of the mean.

Figure 7 The ultrastructure of hippocampal neurons as revealed by TEM.

Notes: P group: i.p. injected with 50 mg/kg propofol, and 40–60 min later, when the righting reflex was recovered, the procedure was repeated one more time; PD₂₅, PD₅₀ and PD₇₅ groups: intraperitoneal injection with 25, 50 and 75 μg/kg Dex, and 30 min later, intervention with propofol; LPD group: intracerebroventricular injection with 25 μg/5 μL LY294002, and 30 min later, intervention with Dex and propofol; TPD group: intraperitoneal injection with 1 mg/kg TDZD-8, and 30 min later, intervention with Dex and propofol. DS₁ and DS₂ groups: intracerebroventricular or intraperitoneal injection with DMSO; F group: intraperitoneal injection with intralipid; L group: intracerebroventricular injection with LY294002; T group: intraperitoneal injection with TDZD-8; Dex group: intraperitoneal injection with Dex. The right picture of each group is the enlarged view of the red box in the left picture. The yellow arrows indicate the presynaptic membrane and vesicles, and the red arrows indicate the postynaptic membrane.
Abbreviations: DMSO, dimethyl sulfoxide; TEM, transmission electron microscopy.

Figure 8 Pretreatment of young rats with Dex mitigated the propofol-attenuated Akt activation in the hippocampus of rats.

Notes: P group: i.p. injected with 50 mg/kg propofol, and 40–60 min later, when the righting reflex was recovered, the procedure was repeated one more time; PD₂₅, PD₅₀ and PD₇₅ groups: intraperitoneal injection with 25, 50 and 75 μg/kg Dex, and 30 min later, intervention with propofol; LPD group: intracerebroventricular injection with 25 μg/5 μL LY294002, and 30 min later, intervention with Dex and propofol; TPD group: intraperitoneal injection with 1 mg/kg TDZD-8, and 30 min later, intervention with Dex and propofol. DS₁ and DS₂ groups: intracerebroventricular or intraperitoneal injection with DMSO; F group: intraperitoneal injection with intralipid; L group: intracerebroventricular injection with LY294002; T group: intraperitoneal injection with TDZD-8; Dex group: intraperitoneal injection with Dex. (A) The relative levels of Akt and GSK3β expression and phosphorylation in the hippocampus of individual rats were determined by Western blot. (B) Data are representative images or are expressed as the mean±SEM of each group (n=5 per group) from three separate experiments. ^*p<0.05 versus NS group; ^#p<0.05 versus P group; ^{^}p<0.05 versus PD₇₅ group; double symbols mean p<0.01.
Abbreviations: Dex, dexmedetomidine; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.