Fig 1.
Schematic diagram of experimental design.
Table 1.
Baseline (pre-cardiac arrest) characteristics of Orexin-A and control groups.
No significant differences in baseline characteristics between animals in CA+Saline (n = 6) and CA+Orexin-A (50 μM) (n = 6).
Table 2.
Hemodynamic parameters at 30 minutes post- ROSC (before drug) and 30 minute post Orexin-A treatment.
No significant differences were observed before treatments. The effects of ORXA (50 μM) or Saline were measured 30 min after the treatment.
Fig 2.
Intranasal orexin-A treatment improves behavioral outcomes after CA.
(A) Neuro Deficit score (NDS) was tested at 4 hrs after ROSC for control (CA+ saline) (n = 6), CA+10μM orexin-A (n = 6), and CA+ 50μM orexin-A (n = 6) treatment groups. (B) Schematic representation of correlations between individual NDS subscores. See S3 Table for correlation coefficients. (C) Subscores of Cluster 1 were improved after treatment with 50 μM ORXA. Values are reported as mean± SEM. Single and double asterisks indicate significant differences from Saline group as a result of LSD post-hoc test with p levels less than 0.025 or 0.0001, respectively. Single and double pound signs indicate significant differences from ORXA-10 group as a result of Bonferroni post-hoc test with p levels less than 0.025 or 0.0001, respectively. See S2 Table for the results of appropriate ANOVAs.
Fig 3.
EEG IQ gamma band (30–50 Hz) for the50μM Orexin-A group (red line, n = 6) and the group of control rats treated with saline (black line, n = 6).
(A) Sub-band EEG gamma power tracing shows that orexin-A leads to improved EEG IQ shortly after intranasal administration in gamma band EEG IQ levels. (B-D) EEG gamma power averaged for periods of baseline recording (B), ROSC (C), and post-treatment (Saline/ ORXA) (D). Data are presented in mean± SEM. BL indicates baseline (pre-anesthesia); CA: asphyxial cardiac arrest; Drug: represents the period after saline or orexin treatment. Asterisk indicates significant between-group differences for the post-drug period, p<0.021. Results of statistical analyses are presented in S4 Table.
Fig 4.
Effects of CA and ORXA treatment (50 μM) on mRNA levels of ORX receptors in different brain structures.
(A) mRNA levels of Orexin receptor 1 and 2 in different brain structures as compared to Cerebellum. (B-C) Effects of CA and ORXA treatment on mRNA levels of ORX receptor-1 (B), and ORX receptor-2 (C). Results are expressed relative to the control group. Asterisks and pound sings indicate significant differences from Sham control or CA+Saline group, respectively, as a result of LSD post-hoc tests with p levels corrected for multiple comparisons, pcor<0.0021 (see S5 Table for results of ANOVA). Probe sequences for RT-PCR are presented in S2 Table.
Fig 5.
Effects of CA and ORXA treatment (50 μM) on mRNA levels of neuroinflammatory markers.
(A) CD11b, (B) iNOS, (C) TNF-α, (D) GFAP, and (E) IL1β. RT-PCR measures were done in brain samples of the prefrontal cortex (PFC), caudal cortex, hippocampus, striatum, hypothalamus, medulla, rest of the brain (ROB), and cerebellum. Asterisks and pound sings indicate significant differences from Sham control or CA+Saline group, respectively, as a result of LSD post-hoc tests with p levels corrected for multiple comparisons, pcor<0.0021 (see S7 Table for results of ANOVA). Probe sequences for RT-PCR are presented in S2 Table.
Fig 6.
Summary of changes in neuroinflammatory markers induced by CA and ORXA treatment.
Raw variables of neuroinflammatory markers were subjected to factor analyses for each of the representative brain structures: (A) the prefrontal cortex, (B) hippocampus, and (C) hypothalamus. Group means ± SEM for Factor scores are shown for CA + Saline and CA + ORXA groups. Inserts in each panel show sets of variables represented by each factor (see S8 Table for Factor loadings). Asterisks indicate significant effect of ORXA, p<0.005, as a result of LSD post-hoc tests (see S9 Table for the ANOVAs of factor scores).