Oxytocin protects neurons from hypoxic-ischemic brain injury by enhancing inhibitory neurotransmission in neonatal rats

Purpose: To study the protective effect of oxytocin on hypoxic-ischemic brain neuron injury in neonatal rats, and the mechanism of action involved. Methods: Hippocampal slices from neonatal SD rats were cultured in oxygen/glucose-deprived (OGD) solution, leading to establishment of hypoxic-ischemic model of hippocampal slices in vitro. The slices were assigned to 3 groups: control (ACSF solution), model (OGD solution), and oxytocin (OGD solution + 1 μM oxytocin). The effect of oxytocin on vertebral neurons in hippocampal CA1 region of HIBD rats was determined using TOPRO-3 staining, while the effects of oxytocin on hypoxic depolarization (AD) and inhibitory postsynaptic currents (iPSCs) were measured by cell patch clamp technique. Results: The fluorescence intensity of vertebral lamina in hippocampal CA1 area of model group was significantly higher than that of control group, while the corresponding value for oxytocin group was significantly lower than that of model group (p < 0.05). The time lapse before occurrence of AD in hippocampal CA1 area was significantly longer in oxytocin group than in model group, while the time lapse before neuronal AD in oxytocin receptor antagonist group was lower than that in oxytocin group. The frequency and amplitude of iPSCs in oxytocin group were markedly higher than the corresponding control values. Conclusion: Oxytocin exerts protective effect against hypoxic-ischemic brain neuronal damage in neonatal rats by regulating the activation of oxytocin receptor and GABA receptor, and inhibiting nerve transmission. These findings may be of benefit in the development of a suitable therapy for HIBD.


INTRODUCTION
Neonatal hypoxic-ischemic brain damage (HIBD) is a severe childhood disability caused by cerebral ischemia and hypoxia during perinatal asphyxia. It manifests mainly in changes in the state of consciousness and muscle tone, and it causes a series of complications such as intellectual retardation and cerebral palsy. Indeed, HIBD has become one of the main causes of neonatal acute death and chronic nervous system injury, resulting in huge economic burden and heavy mental pressure on the affected families and society [1]. Therefore, there is need for in-depth studies on the pathogenesis of HIBD so as to develop newer and more effective treatment methods for the disease.
At present, many researchers believe that multiple mechanisms such as oxidative stress, inflammation and excitatory toxicity are jointly involved in the pathogenesis of HIBD. Excitatory toxicity is closely related to neurotransmitters in the brain, with gamma aminobutyric acid (GABA) as the main neurotransmitter. Signal enhancement is one of the protective mechanisms involved in neuronal ischemia and hypoxia injury. Thus, drugs that enhance GABA signal are expected to be new drugs for the treatment of HIBD [2,3]. The hippocampus is the site most commonly involved in HIBD. Ischemia and hypoxia easily lead to disorders in synaptic transmission in hippocampal neurons, indicating that reducing neuronal damage in the hippocampus may be a promising and new target in the treatment of HIBD [4].
Oxytocin, a physiological hormone produced by the paraventricular nucleus and supraoptic nucleus of the hypothalamus and released in the posterior pituitary gland, has been used in clinical practice for long, due to its potential to induce labor and stimulate milk secretion [5]. Studies have shown that oxytocin produced therapeutic effects on a variety of nervous system diseases. For example, it significantly enhanced inhibitory neurotransmission in hippocampal neurons of young mice [6]. The purpose of this study was to determine the protective effect of oxytocin against hypoxic-ischemic brain neuronal injury in neonatal rats, and to further study the mechanism involved.

Reagents and equipment
The reagents and instruments used in this study, and their sources (in brackets) were: DVOT (

Topro-3 staining
Topro-3 staining solution was prepared with ACSF solution at a volume ratio of 1:2000. After the brain slices were immersed in Krebs solution for 1 h, they were incubated for 20 min, and then placed in the Topro-3 staining solution for 20 min. Thereafter, the slices were soaked in Krebs solution for 10 min to wash away residual dye solution. The brain slices were examined under laser confocal microscopy at excitation wavelength of 639 nm. Slices 20 μm away from the surface were photographed with the same parameters. The fluorescence intensity of CA1 area in each section was calculated.

Whole cell patch clamp technique
The time taken for occurrence of AD was recorded in the current clamp mode. The neuronal membrane potential rose rapidly to 0 mV for AD, and the time of occurrence was the period from the start of oxygen-glucose deprivation, through the occurrence of rapid neuronal depolarization, to the end point. The frequency and amplitude of inhibitory postsynaptic current (IPSCs) were recorded formally and continuously after the cell recording became stable.

Statistics
Measurement data consistent with normal distribution are presented as mean ± SD. Twogroup comparison was done with t-test, while multiple-group comparison was carried out with one-way ANOVA. The SPSS version 21.0 was used for statistical analyses. Statistical significance was assumed at p < 0.05.

Effect of oxytocin on vertebral layer neurons in hippocampal CA1 regions of HIBD rats
Slices of brain tissues from 8 neonatal rats were randomly divided into control group (incubated with normal ACSF fluid), model group (in vitro hypoxic-ischemia model of hippocampal slices incubated with OGD solution), and oxytocin group (OGD solution incubated with 1 μM oxytocin).

Effect of oxytocin on hippocampal CA1 vertebral layer neurons in HIBD rats
The results of TOPRO-3 staining showed that the fluorescence intensity of the vertebral layer in the hippocampal CA1 region was markedly higher in model group than in control group, while the fluorescence intensity of vertebral layer in hippocampal CA1 region of rats in oxytocin group was significantly lower than the corresponding model value. These results are presented in Figure 1 and Figure 2. Table 1 shows that the time lapse before occurrence of AD in the hippocampal CA1 zone of oxytocin rats was significantly higher than that in the model group (p < 0.01), while the times before the occurrence of neuronal AD in the oxytocin receptor antagonist and GABA receptor antagonist group were markedly lower than the corresponding oxytocin values.

Effect of oxytocin on IPSCs of vertebral neurons in hippocampal CA1 region of rats
As shown in Table 2, the frequency and amplitude of IPSCs in the hippocampal CA1 zone of rats were markedly higher in oxytocin group than in the control group, but there was no significant difference in IPSCs decay time between the model and control values.

Effect of different doses of oxytocin on IPSCs of vertebral neurons in hippocampal CA1 region of rats
Oxytocin enhanced IPSCs in the hippocampal CA1 region in a dose-dependent manner, as shown in Table 3

Effect of oxytocin antagonist on IPSCs
The mean frequency of IPSCs was 1.10 ± 0.12 times, and the mean amplitude was 1.00 ± 0.01 times as high as that in the control group when the oxytocin receptor was blocked with 1μM of oxytocin receptor antagonist. These results are shown in Table 4.

DISCUSSION
Hypoxic-ischemic brain damage (HIBD) is a serious disease in the neonatal period, with an incidence of up to 26 % in developing countries. About 20 % of the affected neonates die during the perinatal period, while the survivors suffer neurodevelopmental disorders, epilepsy and learning disabilities, all of which seriously endanger the physical and mental health of children [8]. The pathogenesis of HIBD is not yet fully understood, although it is generally believed to be related to calcium overload, disorders in energy metabolism, and oxygen free radicalinduced damage [9,10]. Therefore, there is need to understand the mechanism involved in the occurrence and development of HIBD, and to evolve a safe and effective treatment strategy for it.
Oxytocin is a neuropeptide hormone synthesized by large cells in the paraventricular nucleus and supraoptic nucleus of the hypothalamus. It is vital for induction of labor and milk secretion. Oxytocin usually acts through a G-protein-coupled receptor to regulate inflammatory response directly related to atherosclerotic cardiovascular disease; it reduces the content of fat and cytokines, and improves glucose tolerance [11,12]. Although oxytocin has not been used in the treatment of heart disease, it has shown positive effects in animal models of atherosclerosis, with prospect for broad clinical applications. In addition, oxytocin can be used as a central neurotransmitter or regulator for learning, memory and maternal behavior. It also acts as a neurotransmitter or regulator in the hippocampus of rats: binding sites have been found in the CA1 region, but the specific mechanism is not fully understood [13]. It has been reported that oxytocin significantly weakened the response of hippocampal neurons to electrical stimulation of sciatic nerve in a dosedependent manner, and also reduced the spontaneous firing frequency of neurons, although there was no obvious dose-dependent relationship in the later effect [14].
The results obtained in the study of the protective effect of oxytocin on hippocampal CA1 neuronal damage in HIBD rats showed that the fluorescence intensity of the vertebral layer in the hippocampal CA1 region of the model group was significantly higher than that of the control group, while the fluorescence intensity of vertebral layer in hippocampal CA1 region in oxytocin group was significantly lower than that in model group. These results suggest that oxytocin can reduce hypoxic-ischemic injury of hippocampal neurons in neonatal rats. Hypoxic depolarization (AD) is one of the early changes in cerebral ischemia. It leads to changes in electrolyte composition within and outside cells, and it is one of the important electrophysiological markers of neuronal death [15,16].
In this study, the effect of oxytocin on time of occurrence of AD was studied. It was found that the time taken for occurrence of AD in the vertebral neurons in hippocampal CA1 region of oxytocin rats was significantly longer than that in the model group, but the occurrence times of neuronal AD in oxytocin receptor antagonist and GABA receptor antagonist were significantly lower than those of oxytocin group, suggesting that oxytocin prolonged the AD time of rat hippocampal vertebral neurons. Gamma aminobutyric acid (GABA) is an excitatory neurotransmitter, and its receptor antagonist completely inhibited the effect of oxytocin on the occurrence time of AD. Therefore, this study has demonstrated that the mechanism of action of oxytocin is related to the effects of its receptor and GABA receptor antagonist.
The frequency and amplitude of IPSCs in hippocampal CA1 region of rats in the oxytocin group were significantly higher than those in control group, indicating that oxytocin increased the frequency and amplitude of IPSCs, but did not change the dynamic characteristics of IPSCs. The results showed that the mean frequency of IPSCs was 1.10 ± 0.12 times that of the control group, and the mean amplitude was 1.00 ± 0.01 times that of the control group. These results suggest that oxytocin could increase the release of GABA by binding to the oxytocin receptor of GABA-mediated neurons. Studies outside China have revealed that in the hippocampal CA1 region of transgenic mice labeled with golden fluorescence, more than 80 % of excitatory neurons expressed oxytocin receptor, while only 19 % of GABA neurons expressed oxytocin receptor [17][18][19].

CONCLUSION
Oxytocin exerted significant protective effect against hypoxic ischemic brain neuronal injury in neonatal rats through a mechanism related to the regulation of oxytocin receptor and GABA receptor activation, thereby inhibiting neurotransmission. These findings may be beneficial in the clinical treatment of HIBD.