Abstract
Objective To detect the expression of FADD in neonatal rats with hypoxic-ischemic brain damage (HIBD) and the regulatory effects of ceftriaxone on the expression of FADD，investigating the neuroprotective effects of ceftriaxone.
Methods A total number of 144 7d-old neonatal SD rats were randomly divided into sham operation group, saline control group and ceftriaxone group, then each group was further divided into 6h subgroup,12h subgroup,24h subgroup,3d subgroup,5d subgroup and 7d subgroup with 8 rats respectively according to observation time.Once the HIBD models were successfully established，in ceftriaxone group，rats were intraperitoneally injected with ceftriaxone (200mg/kg) once a day for 3 consecutive days, and rats of sham operation group and saline control group were intraperitoneally injected with equivalent volume saline in the same way. Neonatal rats were given ceftriaxone or saline at the same time of each day.Then we explore the expression of FADD in neonatal rats by western blot and immunohistochemical staining.The pathological changes were observed by light microscope.
Results There was only a small amount of Expression of FADD in sham operation group；expression of FADD in rats of saline control group was more than that in sham operation group at each time point(
P<0.05)；expression of FADD in rats of ceftriaxone group was less than that in rats of saline control group(
P<0.05)，HE staining showed in sham operation group，the tissue structure and cell arrangement were still clear，nerve cells were arranged in a more orderly and compact way in morphology.In saline control group，the gap between nerve cells in the sick area of brain tissue increased with cells arranging sparsely and obvious edema.Necrosis of nerve cells could be seen in the shape of karyopyknosis and karyorrhexis.The arrangements of nerve cells in ceftriaxone group were organized with increased volume and light edema.
Conclusion Ceftriaxone can inhibit excessive expression of FADD in HIBD neonatal rat brain，which may indicate that ceftriaxone is able to play neuroprotective effect, the potential mechanism is reducing neuron cell apoptosis by inhibiting excessive expression of FADD.
Keywords：ceftriaxone，hypoxic-ischemic brain injury，neonatal rat，FADD，apoptosis
Background
Hypoxic-ischemic brain injury of newborn infants is a common cerebral injury，which results dominantly from perinatal asphyxia and is one of the diseases in relatively high morbidity as well. Considering the relatively high morbidity，it accounts for 25% to 28% in the diseases which lead to cerebral palsy[1]. The brain is extremely vulnerable to injury induced by hypoxia or ischemia. As our knowledge of brain development has increased in the past several decades，so has our recognition that pathophysiological changes profoundly affect the response to medications，at present，there is still no effective interventions for hypoxia-ischemia induced brain damage. Changes of hemodynamics，energy failure，free radical injury，calcium overload，the excitotoxicity of excitatory amino acids，apoptosis and inflammation are regarded to be involved in the pathophysiology in hypoxia-ischemia induced brain injury.
Recent studies demonstrated that apoptosis is responsible for hypoxia-ischemia induced brain injury remarkably[2-4]，which is an important factor in the pathogenesis of hypoxic-ischemic brain injury. Apoptosis is classified as intrinsic apoptosis and extrinsic apoptosis[5,6]. FAS-associated protein with death domain（FADD）is the key adaptor protein transmitting apoptotic signals mediated by the main death receptors(DRs)[7]. It is a kind of bridging protein which links death receptors and signal-transmitting proteins in cytoplasm and is a critical signal-transporter that induce cell apoptosis and is involved in a variety of central nervous system diseases.
β-Lactam antibiotics，ceftriaxone，besides its antimicrobial effects，a pioneering research indicated that it exhibited neuroprotective effects in some neuronal diseases by upregulating significantly glutamate transporter-1（GLT-1）expression[8]. However，a recent study showed that ceftriaxone attenuates hypoxic-ischemic brain injury in neonatal rat by upregulating GLT-1 expression and the authors speculated that reduce of apoptosis may be involved in the neuroprotection of ceftriaxone. In our study，we aim to investigate whether ceftriaxone could modulate the expression of FADD.
1 Methods
1.1 Animals
7d neonatal SD rats were provided by the animals center of Xuzhou Medical University. The pubs were raised with their mothers in individual cages with 12 hrs dark/light cycle and 22-26℃，with free food and water. Seven days after born，neonatal rats were screened for our study by weight ranging from 12-18g.
Neonatal rats model of hypoxic-ischemic brain damage and treatment design
7d neonatal SD rats were randomly divided into sham operation group（group S），normal saline control group（group C）and ceftriaxone group（group CTX）.Each group was further divided into 6 subgroups by observational time at 6h，12h，24h，3d，5d，7d after operation（n=8）.Rats of group S and group C were intraperitoneally injected with 10ml/kg normal saline for 3 consecutive days at the same time point after operation and neonatal rats of group CTX were intraperitoneally injected with ceftriaxone（200mg/kg）with the same methods. Neonatal rat model of hypoxic-ischemic brain injury was created according to Rice method[9]. Rats were inhaled with sevoflurane for anesthesia and then were operated for right carotid artery ligation.After operation，they were sent back to their mothers，resting for 2 hours.The hypoxia model was created by being exposed to 8%N
2/92% O
2（1-2L/min）for 2 hours. Rats of sham operation group were just sham operated without right artery ligation or being exposed to 8%N
2/92% O
2. Finally，they were sent back to their mothers in an environment of 22-26℃temperature with 12hour dark/light cycle with free food and water.
1.2 Brain tissue preparation
Rats were inhaled with sevoflurane for deep anesthesia，in status of deep anesthesia，the blood vessels are completely relaxed. The rats were transcardially perfused with 20ml ice-cold saline followed by 20ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and dipped in 4% paraformaldehyde in 0.1 M phosphate buffer overnight at 4℃，sequentially dipped in 15% sucrose and then 30% sucrose in 0.1 M phosphate buffer until the brains sank for cryoprotection. Brains were then embedded in O.C.T compound for immunohistochemical staining and the freshly removed brains were stored at -80℃ for tissue preparation.
1.3 HE staining and immunohistochemical staining
The pathological changes of brain in each group were observed by light microscope. The right cerebral cortex of each subgroup were fixed in paraformaldehyde at 4℃ for 24 hours.Brain sections were dehydrated with increasing ethanol concentrations and then were embedded in paraffin.Cut fixed brain coronal with a “Brain Blocker”into about 2-mm-thick slices.Put tissue slices in 10 % sucrose overnight (in fridge), in 20 % sucrose a whole day with a little shaking (in room temperature, RT), and in 30 % sucrose overnight (in fridge) and then in 1:1 of 30 % sucrose/OCT solution 4-5 h followed with pure OCT 1-2 h in RT. The brain specimens were fixed with 10% formaldehyde, and cut as 4 μm sections and brain sections were washed with 0.01 M phosphate buffered saline (PBS) for three times.The sections were processed with 3 % hydrogen peroxide for 20 min to block endogenous peroxidases.The brain sections were blocked with 2% goat serum in 0.01 M PBS containing 0.3% Triton X-100 for 1h at room temperature，then incubated at 4℃overnight with antibody against FADD(1:300 dilution, Boaosen, Beijing, China) and developed with secondary antibody. At last，the brain sections were stained with DAB and were observed by light microscope.For negative controls，the antibody against FADD was replaced with PBS.We randomly selected 5 fields from each right cerebral cortex and the immunohistochemical staining score was evaluated according to the average numbers of the positively stained cells using image proplus6.0 system.
1.4 Western blot
100mg fresh tissue of was removed from right cerebral cortex in each group，protein was cleaved by RIPA followed by homogenate and then centrifuged（12000rpm）for 15 minutes at 4℃.The concentration of protein was measured by BCA method. Primary antibody was anti-FADD（1:500，Boaosen，Beijing，China） and β-actin（1:2000，Santa cruz）was used as internal standard. The gray value of the stripe was tested by Image J.
1.5 Statistical analysis
Measurement data were recorded in form of mean ± standard deviation（SD）.Single factor analysis of variance was used in the comparison between groups using SPSS 16.0，
P＜0.05 was considered differences are statistically significant.
2 Results
2.1 HE staining
The pathological changes of the sick area of cerebral cortex were observed with light microscope by HE staining.What we observed showed that in sham operation group，the tissue structure and cell arrangement were still clear，nerve cells arranged in a more orderly and compact morphology.In saline control group，the gap between nerve cells in the sick area of brain tissue increased with cells arranging sparsely and obvious edema.Necrosis of nerve cells could be seen in the shape of karyopyknosis and karyorrhexis.The arrangements of nerve cells in ceftriaxone group were organized with increased volume and light edema（Figure 1）.
Fig.1 HE staining of pathological changes of each group，A: sham operation group，B: normal saline control group，C: ceftriaxone group. Magnification：×400
2.2 Western blot
Western blot results showed that there was only a little expression of FADD in sham operation group at each time point.Expression of FADD in saline control group
was much more than that in sham operation group with statistically significant difference(
P<0.05). Expression of FADD in saline control group was already increased at 6h time point，then ascended，peaking at 3d and then decreased but still remained relatively high；however，expression of FADD in ceftriaxone group was obviously lower than that in saline control group but still higher than that in sham operation with statistically significant difference（
P<0.05）（Table 1，Figure2,3）.
Table 1 FADD expression in each group at each time point by Western blot(n=8,`
x±
s)
|
Group |
6h |
12h |
24h |
3d |
5d |
7d |
|
S |
0.194 ± 0.024 |
0.193 ± 0.024 |
0.201 ± 0.018 |
0.210 ± 0.020 |
0.191 ± 0.020 |
0.204 ± 0.018 |
|
C |
0.305 ± 0.016 |
0.405 ± 0.017 |
0.463 ± 0.023 |
0.639 ± 0.028 |
0.513 ± 0.024 |
0.405 ± 0.020 |
|
CTX |
0.255 ± 0.022 |
0.312 ± 0.021 |
0.351 ± 0.027 |
0.425 ± 0.031 |
0.320 ± 0.031 |
0.280 ± 0.033 |
|
F |
55.677 |
205.842 |
261.750 |
519.450 |
300.800 |
135.755 |
|
P |
<0.05 |
<0.05 |
<0.05 |
<0.05 |
<0.05 |
<0.05 |
S: sham operation group，C: saline control group，CTX: ceftriaxone group. The difference is statistically significant compared with S group，
P<0.05；The difference is statistically significant compared with C group，
P<0.05.
6h 12h 24h 3d 5d 7d
Fig.2 Western blot of FADD in each group in right cerebral cortex，S: sham operation group，C: saline control group，CTX: ceftriaxone group.
Fig.3 Mean OD(FADD/β-Actin)，S: sham operation group，C: saline control group，CTX: ceftriaxone group.*: The difference is statistically significant compared with S group,
P<0.05; #: The difference is statistically significant compared with C group，
P<0.05.
2.3 Immunohistochemical staining
Immunohistochemistry indicated：only a small number of FADD positive cells were detected in sham operation group.In saline control group，the FADD positive cells were much more than those in sham operation group at each time point（
P<0.05）.The FADD positive cells were found already increased at 6h time point，ascended and peaked at 3d and then decreased gradually，but still remained a relatively high level.However，FADD positive cells in ceftriaxone group were less than those in saline group（
P<0.05），but were more than those in sham operation group（
P<0.05）（Table 2，Figure 4）.
Table2 Numbers of FADD positive cells in each group by Immunohistochemical staining(n=8, `
x±
s)
|
Group |
6h |
12h |
24h |
3d |
5d |
7d |
|
S |
495.63±27.34 |
502.63±15.57 |
502.00±23.82 |
505.50±24.35 |
503.75±27.33 |
502.88±20.36 |
|
C |
956.38±59.62 |
1137.75±103.48 |
1366.50±79.91 |
2479.75±162.34 |
2021.13±132.39 |
1567.63±146.63 |
|
CTX |
757.75±53.05 |
891.25±50.83 |
1063.75±92.71 |
1804.88±110.62 |
1422.75±80.89 |
877.13±74.33 |
|
F |
180.13 |
181.80 |
297.02 |
616.70 |
564.95 |
255.17 |
|
P |
<0.05 |
<0.05 |
<0.05 |
<0.05 |
<0.05 |
<0.05 |
S: sham operation group，C: saline control group，CTX: ceftriaxone group. The difference is statistically significant compared with S group，
P<0.05；The difference is statistically significant compared with C group，
P<0.05.
Fig.4 Immunohistochemical staining of each group；A: sham operation group，B: saline control group，C: ceftriaxone group. Magnification：×400.
3 Discussion
Hypoxic-ischemic brain injury is mainly resulted from perinatal asphyxia in newborn infants，part of them often lead to central nervous system sequelae[10]. Children who sustained neonatal hypoxic-ischemic encephalopathy（HIE） without major disability are at increasing risk for long-term intellectual, verbal, and motor deficits[11]. The severity of watershed injury is correlated with later intellectual performance.In children with hypoxic-ischemic encephalopathy，20%-30% of them will develop permanent cerebral palsy[12].Among the pathogenesis which lead to hypoxic-ischemic brain injury，neurocyte apoptosis is an important pathological change.Diagnosis of HIE currently relies on the laboratory diagnosis and imaging examination,but it is often hard to approach early diagnosis.Thus the molecular mechanism of HIE is researched by establishing animal models,which has been gradually applied in clinic[13].In the last decade，apoptosis has been regarded as an important mechanism in neonatal hypoxic-ischemic brain injury.It has been demonstrated that after 48 hours of HI, the number of apoptotic cells was up to the peak level. The best time for treatment might be during 24 to 48 hours after HI, which prohibits the apoptosis and protects brain from HI damage[14].As a result，we choose the time window from 6h to 7d after HI as our observational time window.
Currently，it is recognized that neuronal lessons are led by intrinsic and extrinsic pathway signaling[5].Intrinsic apoptosis is mediated by increased permeability of mitochondrial external membrane and extrinsic apoptosis is mediated by the death receptors which is on cell membrane. FADD is a key adaptor protein which links death receptors and the cytoplasm protein，thereby mediating extrinsic apoptosis. It was reported FADD/caspase-8 signaling is necessary and sufficient for apoptosis of cardiomyocyte subjected to hypoxia[15].Cerebral hypoxia-ischemia led to a strong lateralised upregulation of Fas death receptor in the hippocampus, that peaked six to twelve hours after the insult and was greater on the side of injury[16].It suggested that Fas and FADD may be involved in neuronal apoptosis following hypoxic-ischemic injury to the developing brain. Our study is aimed to investigate whether FADD is involved in the apoptosis in cerebral cortex of hypoxic-ischemic brain injury in neonatal rats and to explore the potential therapy for the kind of injury.
In 2005，Rothstein J D found beta-lactam antibiotics are potent stimulators of GLT-1. Furthermore, this action appears to be mediated through increased transcription of the GLT-1 gene[8]. Animal studies showed that the protein is important for normal excitatory synaptic transmission, while its dysfunction is implicated in acute and chronic neurological disorders, including amyotrophic lateral sclerosis (ALS), stroke, brain tumours and epilepsy. This is the first time that ceftriaxone was found to offer neuroprotection effects.When comes to hypoxic-ischemic brain injury，still others showed that ceftriaxone attenuates hypoxic-ischemic brain injury in neonatal rat，the potential mechanism is ceftriaxone is able to upregulate the expression of GLT-1 thereby reducing the excitotoxicity of excitatory amino acids[17].One study indicated that ceftriaxone modulates apoptosis pathways and oxidative stress in a rat model of neuropathic pain[18]. Involvement of Bax, Bcl2, and caspases 3 and 9, important contributors of programmed cell death (apoptosis)，were determined using western blotting. However，the effects of ceftriaxone on FADD mediated extrinsic apoptosis have not been investigated. So we aimed to investigate whether ceftriaxone is able to reduce FADD expression in neonatal rat with HIBD.
According to our study，we found the expression of FADD in saline control group was more than that in sham operation group(
P＜0.05)，so we conclude that FADD is involved in the pathogenesis of hypox-ischemic brain injury.Both western blot and immunohistochemical staining demonstrated that ceftriaxone reduces expression of FADD in neonatal rat with HIBD.According to HE staining，the pathological lesion was significantly attenuated by ceftriaxone，so we hypothesize ceftriaxone ameliorated neonatal rat HIBD by reducing FADD expression and the potential mechanism is inhibiting FADD mediated extrinsic apoptosis.Our findings may pave the way for novel therapy for hypoxic-ischemic brain injury.
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