Effects of inter-alpha inhibitor proteins on brain injury after exposure of neonatal rats to severe hypoxia-ischemia

Abstract

Hypoxic-ischemic (HI) brain injury is one of the most common neurological problems occurring in premature and full-term infants after perinatal complications. Hypothermia is the only treatment approved for HI encephalopathy in newborns. However, this treatment is only partially protective, cannot be used to treat premature infants, and has limited efficacy to treat severe HI encephalopathy. Inflammation contributes to the evolution of HI brain injury in neonates. Inter-alpha Inhibitor Proteins (IAIPs) are immunomodulatory proteins that have neuroprotective properties after exposure to moderate HI in neonatal rats. The objective of the current study was to determine the neuroprotective efficacy of treatment with IAIPs starting immediately after or with a delay of one hour after exposure to severe HI of 120 min duration. One hundred and forty-six 7-day-old rat pups were randomized to sham control, HI and immediate treatment with IAIPs (60 mg/kg) or placebo (PL), and sham, HI and delayed treatment with IAIPs or PL. IAIPs or PL were given at zero, 24, and 48 h after HI or 1, 24 and 48 h after HI. Total brain infarct volume was determined 72 h after exposure to HI. Treatment with IAIPs immediately after HI decreased (P < 0.05) infarct volumes by 58.0% and 44.5% in male and female neonatal rats, respectively. Delayed treatment with IAIPs after HI decreased (P < 0.05) infarct volumes by 23.7% in male, but not in female rats. We conclude that IAIPs exert neuroprotective effects even after exposure to severe HI in neonatal rats and appear to exhibit some sex-related differential effects.

Keywords: Brain injury; Hypoxia-ischemia; Inter-alpha inhibitor proteins; Neonatal rats; Neuroprotection; Sex.

Figure 1

(A) Schematic for immediate treatment with IAIPs. The pups were returned to the dams for 1.5 to 3 hours after right common carotid artery ligation. Thereafter, the pups were exposed to 8% oxygen with balanced nitrogen for 120 minutes at a constant temperature of 36 °C. Sixty mg/kg of human blood derived IAIPs or placebo were given intraperitoneally (I.P.) immediately (zero), 24 and 48 hours after termination of hypoxia. Body weight was measured before surgery, immediately (zero), 24, 48 and 72 hours after hypoxia. Seventy-two hours after hypoxia, necropsy was performed, and brain weight obtained. (B) Schematic for the delayed treatment with IAIPs. The procedures were the same as for the schematic in A except that IAIPs or placebo were given 1, 24 and 48 hours after the termination of hypoxia. Body weight was measured before surgery, 1, 24, 48 and 72 hours after hypoxia. Seventy-two hours after hypoxia, necropsy was performed, and brain weight obtained. The − / + signs indicate the time (hours) before/after the end of hypoxia (0 hour). IAIPs = Inter-Alpha Inhibitor proteins, PBS = phosphate buffered saline.

Figure 2

Body weight gain (%) and brain weights (g) of the Sham, HI-PL and HI-IAIP groups after immediate treatment with IAIPs or placebo in neonatal rats exposed to HI related brain injury. (A) Percent body weight gain on the y-axis plotted against study time in hours on the x-axis in the total cohort of males + females, males, and females. Body weight gain (%) was lower in the HI-PL and HI-IAIP total cohort compared with the Sham group during the study periods. Body weight gain (%) was greater in the HI-IAIP total cohort compared with the HI-PL group during the studies. Body weight gain (%) was lower in the HI-PL and HI-IAIP males than in the Sham group during the study periods. The body weight gain (%) was greater in the HI-IAIP compared with HI-PL males but not in the females. Sham: male n = 10, female n = 10; HI-PL: male n = 8, female n = 8; HI-IAIP: male n = 11, female n = 12. Values are mean ± SD. (B) Brain weights in the total cohort of males plus females, males, and females plotted as dot plots showing values as mean ± SD. Brain weights (g) were lower in the HI-PL and HI-IAIP cohort and in the males than in the Sham group, but not in the females. Sham: male n = 10, female n = 10; HI-PL: male n = 8, female n = 8; HI-IAIP: male n = 11, female n = 12. *P < 0.05.

Figure 3

Infarct volume measurement after immediate treatment with IAIPs. (A) Representative images of brain sections stained with cresyl violet 72 hours after exposure to severe HI brain injury. The “I” designates the hemisphere ipsilateral to the carotid artery ligation. Scale bar = 3 mm. Compared to the HI-PL hemisphere, the damaged area in both males (top row) and females (bottom row) were reduced after immediate treatment with IAIPs. (B) Percent infarct volume plotted on the y-axis for the Sham, HI-PL, and HI-IAIP groups on the x-axis for the cohort, males, and females. Values are mean ± SD. *P < 0.05. Sham: male n = 9, female n = 9; HI-PL: male n = 6, female n = 7; HI-IAIP: male n = 11, female n = 12.

Figure 4

Body weight gain (%) and brain weights (g) of the Sham, HI-PL and HI-IAIP groups after delayed treatment with IAIPs or placebo in neonatal rats exposed to HI related brain injury. (A) Percent body weight gain on the y-axis plotted against study time in hours on the x-axis for the cohort, males, and females. Body weight gain (%) was lower in the HI-PL and HI-IAIP total cohort, males and females compared with the Sham group during the study periods. Sham: male n = 19, female n = 21; HI-PL: male n = 12, female n = 12; HI-IAIP: male n = 11, female n = 12. Values are mean ± SD. (B) Brain weights of the cohort, males, and females plotted as dot plots showing values as mean ± SD. Brain weights (g) were lower in the HI-PL and HI-IAIP cohort and in the HI-PL males, and in the HI-PL and HI-IAIP females compared with the Sham group. Sham: male n = 19, female n = 21; HI-PL: male n = 12, female n = 12; HI-IAIP: male n = 11, female n = 12. *P < 0.05.

Figure 5

Infarct volume measurement after delayed IAIP treatment. (A) Representative images of brain sections stained with cresyl violet three days after HI brain injury for males (top row) and females (bottom row). The “I” designates the hemisphere ipsilateral to the carotid artery ligation. Scale bar = 3 mm. Compared to the HI-PL hemisphere, the infarct area in males was reduced after delayed IAIP treatment. (B) Percent infarct plotted on the y-axis for the Sham, HI-PL, and HI-IAIP groups on the x-axis for cohort, males, and females. Values are mean ± SD. *P < 0.05. Sham: male n = 18, female n = 19; HI-PL: male n = 10, female n = 12; HI-IAIP: male n = 10, female n = 11.