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Comparison of the Effects of Citicoline and Piracetam on Hypoxic-ischemic Brain Damage in Neonatal Rabbits

Objectives Perinatal hypoxic-ischemic brain injuries have been a major cause of mortality and neurodevelopmental morbidities in newborns. Citicoline and Piracetam have been used as nootropic agents in a number of studies. In this investigation, we aimed to determine the effects of these agents solely and in combination in hypoxic-ischemic brain damage in rabbit neonates. Materials & Methods Hypoxic-ischemic brain damage was induced by the occlusion of both uterine arteries of dams for eight minutes. The subjects were randomly divided into five groups as follows (n=6 per group): control group without hypoxia (C1), control group with hypoxic-ischemic damage (C2), the third group (P) received Piracetam (100 mg/kg), the fourth group (T) administered with Citicoline (250 mg/kg), and the fifth (PT) received both. The preventive effects of the two drugs on hypoxic-ischemic brain damage were microscopically investigated by the rates of damage to the hippocampus. Results Neuronal destruction rates in C1, C2, P, T, and PT were 4%, 45%, 37.5%, 12.5% (P=0.01 vs. C2), and 20% (P=0.03 vs. C2), respectively. The total means of hypoxic-ischemic damage, cell edema, neuronal degeneration, and eosinophilic degeneration were lower in the T group compared to C2 (P<0.05). Conclusion According to our results and previous findings, Citicoline as a treatment for hypoxic-ischemic brain injuries could be beneficial, and it has priority over neuroprotective agents like Piracetam. Moreover, the combination of Citicoline and Piracetam showed no superior effect in contrast with Citicoline alone. However, experimental studies on larger populations and clinical trials are highly suggested.


Introduction
Hypoxic-ischemic (HI) brain injury is a condition that occurs when the brain components confront hypoxia, ischemia, and cytotoxicity (1). This disorder typically arises after cardiac arrest, respiratory arrest, incomplete suffocations, poisonous gas exposure, and perinatal asphyxia.
Hypoxic-ischemic brain injury frequently produces symptoms such as disturbance of sensorimotor functions, seizure, and impairment of cognitive and emotional tasks (2, 3). The sequelae of the damage depend on the underlying mechanism, the severity, time course, and the duration of oxygen insufficiency and absence of bloodstream (1).
Perinatal HI brain injuries, such as asphyxia, have been a major cause of mortality in newborn infants and chronic neurodevelopmental deficits in survivors (4). Investigations have reported that about 4 million newborns are affected by moderate to severe asphyxia in the developing countries, 20% of whom suffer from its sequelae, for example, HI encephalopathy (5, 6). However, hypoxic-ischemic encephalopathy (HIE) has a mortality rate of 60%, and its treatment is now limited to intensive supportive care (7-9).  (12)(13)(14). Since a few decades ago, Piracetam has been advertised as a nootropic agent in several countries; the drug also has been used for the treatment of cognitive disorders, myoclonus, dyslexia, and vertigo (15,16). The exact mechanism of Piracetam is yet to be cleared; however, there is growing evidence proving that the agent's properties rely on the restoration of the cell membrane fluidity (15). Fixed dosage combination of Citocoline and Piracetam (500 mg and 800 mg) is also available in some markets and is used for memory enhancement, neurological and cognitive disorders, Parkinsonism, and Alzheimer's disease (17).
Considering the magnitude of the problem of HI brain injuries and the lack of accurate medication for the condition, it is appropriate for health-care professionals to give priority to the

Animal models
Rabbits were chosen as the subjects of this experiment mainly due to similarities of their biology with human beings, the high rate of pregnancy, and the high numbers of newborns.
Thirty pregnant New Zealand white rabbits, provided from the Animal Laboratory of Shiraz University of Medical Sciences, were randomly selected for the study. They were kept at 25±2°C temperature and in 60% humidity and regular light/dark cycle with adequate food and water for one week before the experiments.
The models were randomly divided into five groups (n=6). The control group without any hypoxiaischemia (C1) and the control group with hypoxiaischemia (C2), which were killed immediately after inducing hypoxia, and their brain sections were

Induction of hypoxic-ischemic brain damage
Then, the clips were removed for 10 minutes to allow cerebral reperfusion while they were exposed to room air (21% oxygen), and then the fetuses were expulsed. The newborn rabbits were clinically evaluated to ensure their viability. (18)

Assessments
Alive neonates were sacrificed after 10 minutes of exposure to a high dose of pentobarbitone (1 g, Specia™, France). The heads of the neonates were immersion fixed for two weeks in 10% paraformaldehyde in 0.1 M phosphate buffer.
Therefore, the brains were systematically cut into 4-mm thick coronal blocks of the hippocampus and were embedded in paraffin and sectioned at 5-micron range with a vibrating microtome. To assess the destruction rate and to determine the

Statistical analysis
Histopathological scores were designed as median range and mean on graphs by GraphPad Prism software; also, they were analyzed using a nonparametric Mann-Whitney U test by SPSS software (version 21.0, Chicago, IL, USA). A P-value of less than 0.05 was considered statistically significant.

Results
Neuronal destruction in the C1 group was about 4%, and the C2 group showed about 45% damage.
In the T group, the rate of destruction was about 12.5%, which was significantly lower than that of C2. The destruction rate of the P group was near 37.5%, and the rate of destruction in the PT group was about 20-25% (Table 1). The results indicated that administrating intravenous Citicoline alone or in combination with Piracetam can decrease cell death in comparison to the untreated hypoxicischemic brain-injured control group (P ≤ 0.05).
Citocoline could also noticeably decline brain Table 1. Intensity of the hypoxic-ischemic damage in the hippocampus of the rabbits of control group without any hypoxia (C1), control group with hypoxia (C2), experimental group with hypoxia treated with Piracetam (P), experimental group with hypoxia treated with Citicoline (T) and the experimental group with hypoxia treated with Piracetam and Citicoline (PT). Although the PT group showed a considerably lower damage of neuronal degeneration, the total average of hypoxic-ischemic damage in the P group and PT group was 36.3±4.42% and 27.9±10.94, respectively, which were not significantly different from that of the C2 group. Moreover, there was no significant reduction in the brain edema of the P and PT groups, and the degrees of brain edema were approximately near that of the control group (P > 0.05).

Discussion
Hypoxic-ischemic brain injury in general, and specifically, neonatal encephalopathy due to perinatal HI conditions are associates with high mortality, morbidity, and chronic lifelong disabilities (18). Currently, the treatment of HIE in newborns is limited to intensive supportive care, such as the reduction of whole-body temperature and brain temperature (9). However, reports have suggested that cell therapy and some drugs such as Alluporinol and Astragalus could be used in the case of perinatal HI brain damage (19)(20)(21).
Our study showed that rabbit neonates who suffered from HI brain damage and were treated with Citocoline had a significantly lower degeneration and edema of neuronal cells besides lower total average HI destruction of the brain in comparison with the ones that did not receive the medication.
The results were consistent with the findings of a prior study performed by Rao et al., which showed that the drug had protective efficacy against bloodbrain barrier (BBB) dysfunction in the forebrain of gerbils. Başkaya et. al reported a significant reduction in brain edema and BBB breakdown after using the agent in rats affected by traumatic brain injury (22). Similar perinatal asphyxia models of rats indicated that the drug had a dose-dependent neuroprotective effect (23). Another study also supported the hypothesis of prescribing Citocoline for reducing the brain lesion growth produced by ischemic strokes by using diffusion-weighted magnetic resonance imaging (24). Investigations