Effects of ephedrine on expression of Nogo-A and synaptophysin in neonatal rats following hypoxic-ischemic brain damage*★

Siyuan Chen1, Nong Xiao1, Xiaoping Zhang2

1Department of Rehabilitation Center, Children’s Hospital of Chongqing Medical University, Chongqing  400014, China
2Research Institute of Peadiatrics, Children’s Hospital of Chongqing Medical University, Chongqing  400014, China

Siyuan Chen★, Studying for master’s degree, Department of Rehabilitation Center, Children’s Hospital of Chongqing Medical University, Chongqing   400014, China

Corresponding author: Nong Xiao, Doctor, Professor, Master’s supervisor, Department of Rehabilitation Center, Children’s Hospital of Chongqing Medical University, Chongqing  400014, China   
xiaonongwl@163.com

Supported by: the Scientific Research Program of Health Bureau of Chongqing City, No. [2007]1-07-2-153*

Abstract
BACKGROUND: Central nervous system axons regenerate poorly following neonatal hy-poxic-ischemic brain damage (HIBD), partly due to inhibitors, such as Nogo-A. Very few studies have addressed the regulation of Nogo-A in neonatal rats following HIBD. However, numerous studies have shown that ephedrine accelerates neuronal remodeling and promotes recovery of neural function in neonatal rats following HIBD.
OBJECTIVE: To investigate the effects of ephedrine on expression of Nogo-A and synaptophysin in brain tissues of neonatal rats following HIBD.
DESIGN, TIME AND SETTING: A completely randomized, controlled study was performed at the Immunohistochemistry Laboratory of the Research Institute of Pediatrics, Children’s Hospital of Chongqing Medical University from August 2008 to March 2009.
MATERIALS: Ephedrine hydrochloride (Chifeng Pharmaceutical Group, China), rabbit anti-Nogo-A polyclonal antibody (Abcam, UK), and rabbit anti-synaptophysin polyclonal antibody (Lab Vision, USA) were used in this study.
METHODS: A total of 96 healthy, neonatal, Sprague Dawley rats were randomly assigned to three groups (n = 32): sham operation, HIBD, and ephedrine. The HIBD model was established by per-manent occlusion of the left common carotid artery, followed by 2 hours of hypoxia (8% oxygen and 92% nitrogen). In the sham operation group, the left common carotid artery was exposed, but was not ligated or subjected to hypoxia. Rats in the ephedrine group were intraperitoneally injected with ephedrine immediately following HIBD, with 1.5 mg/kg each time. Rats in the sham operation and HIBD groups were injected with an equal volume of saline. All neonatal rats were treated once daily for 7 days.
MAIN OUTCOME MEASURES: Histopathological damage to the cortex and hippocampus was determined by hematoxylin-eosin staining. Expression of Nogo-A and synaptophysin was detected using immunohistochemical staining.
RESULTS: Neuronal degeneration and edema were observed in the hypoxic-ischemic cortex and hippocampus by hematoxylin-eosin staining. Compared with the sham operation group, the levels of Nogo-A significantly increased in the HIBD group at various time points (P < 0.01). Nogo-A ex-pression was significantly reduced in the ephedrine group compared with the HIBD group (P < 0.01). Synaptophysin expression was significantly decreased in the hypoxic-ischemic cortex, compared with the sham operation group (P < 0.01). Synaptophysin levels were significantly increased in the ephedrine group, compared with the HIBD group (P < 0.01).
CONCLUSION: Altered Nogo-A expression was associated with inversely altered synaptophysin expression. The use of ephedrine normalized expression levels of Nogo-A and synaptophysin fol-lowing HIBD.
Key Words: hypoxic-ischemic brain damage; ephedrine; Nogo-A; synaptophysin; brain injury; tra-ditional Chinese herbal medicine

INTRODUCTION
  
The absence of neuronal plasticity following hypoxic-ischemic brain damage (HIBD) can be attributed to several factors, including glial scars, lack of neurotrophic factors, and growth-inhibitory molecules[1]. Nogo-A is one of the most powerful growth inhibitors among the myelin-associated inhibitors[2]. Because Nogo-A plays a pivotal role in the regeneration of neurites in the central nervous system, expression of Nogo-A following brain damage is of great interest. Previous studies[3] have reported that Nogo-A is slightly upregulated in neurons near the injury site following traumatic lesions to the cortex or spinal cord in adult rats. Additionally, Nogo-A expression is significantly increased following transient global ischemia in adult rats[4].
Synaptophysin is an important regulator of axonal outgrowth, which is normally expressed in growing axon terminals and membranes of presynaptic vesicles[5], and is decreased in vulnerable brain legions, such
 
as the hippocampus, following experimental brain injury[6-7]. Ephedrine is a principle compound isolated from the stems of Ephedra sinica, a traditional Chinese herbal medicine. Because it is an indirect sympathomimetic agent, ephedrine exhibits many pharmacological effects[8]. Previous studies have shown that ephedrine exhibits protective effects on newborn rats suffering from HIBD by decreasing neuronal loss and promoting neurotrophin expression[9]. However, it remains to be determined whether ephedrine could upregulate regenerative events by modulating expression of Nogo-A and synaptophysin.
The present study was designed to investigate the effects of HIBD on growth-inhibitory molecules, such as Nogo-A, and the capacity of ephedrine to modulate neuronal injury. In addition, to determine the potential molecular response to Nogo-A inhibition, expression of synaptophysin, an indicator of synaptic growth, was measured.

MATERIALS AND METHODS

Design
A completely randomized, controlled study.
Time and setting
The experiment was performed at the Immunohistochemistry Laboratory of the Research Institute of pediatrics, Children’s Hospital of Chongqing Medical University from August 2008 to March 2009.
Materials
Experimental animals
Sprague Dawley rats with dated pregnancies were maintained at the Animal Center of the Children’s Hospital of Chongqing Medical University (Permit No. SYXK (Yu) 20040001) and housed in individual cages with free access to water and chow. A total of 96 spontaneously delivered offspring were reared with their dams. Protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[10].
Drug product
(1R, 2S)-2-methylamino-1-phenyl-1-propanol ephedrine hydrochloride[11] (batch No. 420325) was purchased from Chifeng Pharmaceutical Group, China. Concentration: 0.225 g/L; molecular formula: C10H15NO?HCl; chemical structural formula:

Equipment and reagents used in this study are as follows:

Methods
Experimental grouping and establishment of hypoxia-ischemia models
A total of 96 Sprague Dawley, neonatal rats, weighing 12–15 g, were randomly assigned to three groups (n = 32 in each group). Rats in the sham operation and HIBD groups were intraperitoneally injected with normal saline. Rats in the ephedrine intervention group were intraperitoneally administered ephedrine at a dose of  1.5 mg/kg[12]. All rat pups were treated once daily for 7 days.
Cerebral hypoxia-ischemia was induced by a modified previously described method[9, 13]. The pups were anesthetized with 10% chloral hydrate (3.0 mL/kg). The neck was incised in the midline, and the left common carotid artery was permanently ligated with a 5-0 surgical silk. The duration of anesthesia and surgery did not exceed 5 minutes per pup. A heat lamp was used during the procedure to maintain body temperature at 37 °C. Following a 2-hour recovery and feeding period, the animals were exposed to 2 hours of hypoxia (8% O2 and 92% N2) by placing them in airtight containers partially submerged in a 37 °C water bath to maintain a constant thermal environment. In the sham operation group, the left common carotid artery was exposed, but was not ligated or subjected to hypoxia.
Sample preparation and morphological analyses
At 1, 2, 3, and 4 weeks after HIBD, as well as for the sham controls, rats were intraperitoneally anesthetized with an overdose of 10% chloral hydrate. The thorax was opened, and the left ventricle was perfused with  0.1 mol/L phosphate buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.4). Following perfusion, the brains were removed and post-fixed in the same fixative for 3 days. The paraffin-fixed brains were serially sectioned into 6-μm coronal slices and stored at -4 °C until further use. For routine histological studies, paraffin sections were stained with hematoxylin and eosin[14].
Immunohistochemistry
Paraffin-embedded sections were used for immunohistochemistry according to previously described methods[15]. Briefly, sections were deparaffinized in dimethyl benzene and rehydrated through 100%–70% graded ethanol, followed by distilled water, and incubated in 3% hydrogen peroxide (H2O2) for 20 minutes to block endogenous peroxidase. Following microwave antigen retrieval, and blocking in goat serum for 30 minutes at 37 °C, the sections were incubated with primary antibodies (rabbit anti-Nogo-A 1: 400 and rabbit anti-synaptophysin 1: 200) at 4 °C overnight. Following rinsing with PBS, the sections were incubated with goat anti-rabbit IgG for 30 minutes, and then incubated with horseradish peroxidase-labeled streptavidin for 30 minutes at 37 °C. Peroxidase activity was revealed by dipping the sections in a mixture containing 0.05% 3, 3′-diaminobenzidine and 0.03% H2O2 for 5 minutes. Sections were then air-dried, dehydrated, and coverslipped. Five visual fields from the hippocampus and cortex were chosen from each section. Images from the slides were obtained using a computer-assisted image analyzer system. A control serum, instead of the primary antibody, was applied to other sections of the same brain samples and served as the negative control.
Main outcome measures
Histopathological damage to the cortex and hippocampus was determined by hematoxylin-eosin staining. Expression of Nogo-A and synaptophysin was detected using immunohistochemical staining.
Statistical analysis
All data were analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA). The results were expressed as Mean ± SD. The differences among groups were assessed using one-way analysis of variance, followed by Student-Newman-Keuls test for multiple comparisons. A value of P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of experimental animals
A total of 96 neonatal rats were included in the final analysis, without any loss.
Pathological changes in the cerebral cortex and hippocampus of neonatal rats
Seven days after HIBD injury, rat brains from various groups were paraffin-embedded, sectioned, and pathological damage was observed with hematoxylin-eosin staining. The left hemisphere exhibited cellular shrinkage and darkening extending throughout the hypoxic-ischemic hemisphere. Injured neurons, with twisted, axonal processes, and cell loss were observed in the hippocampus and cerebral cortex. There was no neuronal damage or edema observed in the sham controls (Figure 1A). Moderate cell edema and diffuse neuronal degeneration were the most obvious changes in the HIBD group (Figure 1B). These histopathological alterations were reduced in the ephedrine group (Figure 1C).
Nogo-A expression in the cerebral cortex of neonatal rats
In the sham operation group, there were few Nogo-A-positive cells at the various time points. The number of cells at each time point in the hypoxic-ischemic cortex was significantly greater than the sham operation group (P < 0.01). Nogo-A expression was increased at 1 week, peaked at 3 weeks, and remained a high level at 4 weeks following HIBD. Following ephedrine treatment, even though the number of cells at each time point remained higher than the sham operation group (P < 0.01), the number of cells was significantly reduced compared with the HIBD group at 2, 3, and 4 weeks following HIBD (P < 0.01; Table 1, Figure 2).

Synaptophysin expression in the hippocampus of neonatal rats
The findings from the present study demonstrated significantly reduced synaptophysin protein expression in the cortex at 1, 2, 3, and 4 weeks following hypoxia-ischemia damage, compared with the sham operation group (P < 0.01). Furthermore, ephedrine likely exerted protective effects, because synaptophysin levels were significantly increased in the ephedrine group at 1, 2, and 3 weeks after HIBD (P < 0.01). Results showed that HIBD decreased synaptophysin levels in the hippocampus. This effect was abolished by ephedrine (Table 2, Figure 3). Positive cells were not detected in the negative control samples.

DISCUSSION

HIBD is a significant cause of neurodevelopmental impairment and disability[16]. However, there are no reliable treatment strategies to mitigate neurological injury and resulted impairments in the clinical setting[17]. As a model of human brain development, the postnatal day 7 (P7) mouse, or rat, approximates a 34–36 week old fetus. This developmental age was used, because this age represents the middle of the brain growth spurt period in rodents[18]. In the present study, neuronal degeneration and edema were observed in the ischemic cortex with hematoxylin-eosin staining, which demonstrated that model establishment was successful.

Nogo-A is one of the major myelin growth inhibitory proteins[19]. It is possible that elevated Nogo-A may retard the post-injury regenerative attempts to repair damaged axonal circuitry[20]. The present data indicated that Nogo-A expression was long-lasting and remained highly expressed even at 4 weeks after HIBD. Nogo-A expression around the axons could explain the lack of axonal regeneration. Increased Nogo-A levels were associated with decreased synaptophysin expression, which suggested that Nogo-A could reduce the capacity for synaptic plasticity and neurite outgrowth. Immunohistochemistry results shown that the use of ephedrine reduced Nogo-A expression and elevated hippocampal levels of synaptophysin, which may be beneficial to neuronal regeneration following brain injury[21].
Increased Nogo-A protein expression may be a critical obstacle for nerve regeneration following HIBD. The ap-plication of ephedrine following HIBD normalized levels of Nogo-A and synaptophysin. The overall results em-phasized the potential therapeutic action of ephedrine to elevate the capacity for plasticity and repair in the injured brain. Future studies should, therefore, focus on the de-velopment of strategies to reduce growth-inhibitory molecules, and increase growth-promoting substrates, to stimulate brain plasticity following HIBD.

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 (Edited by Chen X/Qiu Y/Song LP)