Apelin inhibits motor neuron apoptosis in the anterior horn following acute spinal cord and sciatic nerve injuries☆

Zhiyue Li, Weiguo Wang, Qun Zhao, Weiquan Ning, Bin Yang, Suling Zhang, Siyin Feng

Department of Orthopedics, Third Xiangya Hospital, Central South University, Changsha 410013, Hunan Province, China

INTRODUCTION

Apelin is an important peptide and is the endogenous ligand for the G-protein-coupled putative receptor protein that is related to the angiotensin receptor AT1 (APJ). In humans, apelin is widely expressed in various organs such as the spinal cord, corpus callosum,amygdala, substantia nigra and pituitary gland[1-3]. The APJ receptor is found in the white matter of the spinal cord and in the neurons of the brain, and is involved in the physiological activities of various systems[4].

Studies have investigated the changes that occur in the apelin/APJ system during pathologic states or after peripheral nerve injury. However, few studies have focused on the effects of the apelin/APJ system on acute spinal cord injury (SCI)and sciatic nerve injury. To further understand the role of apelin in SCI and peripheral nerve injury, the present study established a rat model of acute SCI and sciatic nerve injury to investigate apelin expression in the injured spinal cord after sciatic nerve ligation. In addition, we aimed to determine the effects of apelin protein injection at the sciatic nerve stump on anterior horn cell apoptosis.

RESULTS

Apelin protein expression in the normal and injured spinal cord at L4-L6

Sprague-Dawley rats were assigned to normal and SCI groups. The SCI model was established and spinal cord samples at L4-6were harvested for immunohistochemical staining. Results showed that apelin was expressed in the entire spinal cord. Apelin-positive cells stained brown/yellow,mainly in the cytoplasm, when compared with cells in normal spinal cord tissue. Terminal deoxynucleotidyl transferase dUTP nick end labeled (TUNEL)-positive cells, i.e.cells with dark blue nuclei, that were apelin-positive stained brown, and conduction fibers in white matter also stained brow. No evident apelin expression was observed in glial cells or intercellular organelles (Figure 1A).

In the SCI group, the yellow staining of the cytoplasm and nerve fibers deepened at 2 hours post injury, and the intercellular organelles also stained yellow. Apelin expression was significantly greater when compared with the normal group (P ＜ 0.05; Figure 1B, Table 1).

At 6 hours after SCI, some neurons presented with swollen cell bodies, weakened staining, and broken nuclei. No blue staining was detected. At 12 hours post injury, some apoptotic cells were observed, manifested by vacuolar changes and weakened staining. At 24 hours post injury, staining was further weakened, and cell bodies of most neurons presented vacuolar changes,with no yellow staining (Figure 1C).

Figure 1 Immunohistochemical staining of apelin protein expression in the normal and injured spinal cord at L4-6(× 400). There is evident apelin expression in normal spinal cord, an increase in apelin expression at 2 hours and decrease at 24 hours with lightest staining.

At 48 hours post injury, some cells were shrunken or broken, and cells exhibited light staining. The residual fragments of dead cells varied in appearance. At 72 hours, neurons were not observed. No intact cell bodies were observed. Apelin expression in the SCI group significantly increased at 2 hours post injury and gradually decreased with time to significantly lower levels than in the normal group (P ＜ 0.05; Table 1).

Table 1 Changes in apelin expression in the normal and injured spinal cord at different time points

Apelin expression changes in the injured spinal cord at L4-6 following sciatic nerve ligation (SNL)

In the SNL group, spinal cord neurons did not stain yellow after 1 day, but nerve fibers in the white and grey matter darkly labeled yellow; at 3 days after SNL, yellow staining was dark, and blood capillary hyperplasia was observed; at 7 days after SNL, staining of nerve fibers was reduced compared with tissue 3 days post SNL.

Staining was further reduced at 14 days, with significant blood capillary hyperplasia; at 21 days, staining was very light, but remained darker than the normal group. Posterior horn apelin expression was greater than that observed in the anterior horn at each time point (Figure 2,supplementary Figures 1-5 online).

Apelin expression in the spinal cord after SNL was significantly greater than the normal group (P ＜ 0.05).

Apelin was mainly expressed in nerve fibers of the posterior horn, peaked at 3 days, gradually decreased at 7, 14 and 21 days, and was weak at 21 days, but remained greater than the normal group (P ＜ 0.05;Table 2).

Figure 2 Apelin expression in the posterior horn at L4-6 following sciatic nerve ligation (immunohistochemical staining, × 400). Strong apelin expression is evident in the posterior horn of the spinal cord 1 day after injury when compared to the normal group. Strong staining is evident at 3 days followed by a decrease in expression at 7 days.

Table 2 Changes in apelin expression in the spinal cord at L4-6 at different time points following sciatic nerve ligation

Apelin expression and apoptosis in the injured spinal cord at L4-6 following sciatic nerve disconnection

The sciatic nerves were disconnected and sutured. The apelin group was injected with apelin protein at L4-6, while the control group was injected with 0.9% (w/v) normal saline.

Immunohistochemistry showed that the yellow staining of the neuronal cytoplasm and nerve fibers was more evident at 4 hours after sciatic nerve injury, as was the staining of the intercellular organelles.

Staining in the apelin group was reduced at 8, 24 and 72 hours, and 7 and 14 days after injection. The majority of neurons presented vacuolar cell bodies, with no yellow staining. The residual fragments of dead cells varied in appearance (Figure 3).

Figure 3 Apelin expression in the injured spinal cord at L4-6 following sciatic nerve disconnection (immunohistochemical staining, × 400). (A) Yellow staining of the neuronal cytoplasm and nerve fibers was more evident at 4 hours after sciatic nerve injury, and yellow staining was observed in intracellular organelles. Staining in the apelin group was reduced at 8 hours (B) and 72 hours (C) after apelin protein injection, and the majority of neurons presented with vacuolar cell bodies, with no yellow staining.

Apoptosis was detected using the TUNEL method. Results showed that a few TUNEL-positive cells were observed in the anterior horn at 4 hours after sciatic nerve injury; typical apoptotic cells were observed at 8 hours,manifested by karyopyknosis, condensed chromatin or irregular fragments with yellow fluorescence. At 24 hours,the number of apoptotic cells increased, and peaked, but fewer anterior horn apoptotic neurons were observed when compared with the control group. At 72 hours-7 days, the number of apoptotic neurons and glial cells gradually decreased, but blood capillary hyperplasia was observed. Few apoptotic cells were detected for up to 14 days (Figure 4A, supplementary Figures 6, 7 online).

After apelin injection for 4 hours, apoptosis was observed in glial cells in the grey and white matter of the spinal cord and motor neurons in the anterior horn, with dark brown/yellow stained particles in the nuclei; apoptotic bodies were not observed. At 8-24 hours, the number of TUNEL-positive cells increased in the control group, along with a large amount of anterior horn motor neurons and glial cells with karyopyknosis, condensed chromatin and the absence of Nissl bodies. At 72 hours, the number of apoptotic cells peaked, and gradually decreased thereafter.

Up to 7 days, the number of apoptotic cells was similar to that at 8 hours, but the extent of apoptosis was ameliorated,most likely due to non-typical apoptosis. At 14 days,TUNEL-labeled motor neurons were observed in the anterior horn (Figure 4B, supplementary Figures 8, 9 online).

Figure 4 Anterior horn neuronal apoptosis at 7 days after sciatic nerve injury (transferase dUTP nick end labeled staining, × 400). (A) Apoptotic neurons and glial cells in the anterior horn of the control group gradually decreased, and non-typical apoptotic cells at different stages were observed; there was a reduction in the number of glial cells,which was accompanied by blood capillary hyperplasia.(B) Neuronal apoptosis in the anterior horn was ameliorated in the apelin group.

The number of apoptotic neurons in the anterior horn was significantly decreased in the apelin group compared with the control group (P ＜ 0.01; Table 3).

Table 3 Number of apoptotic motor neurons in the anterior horn after sciatic nerve injury by TUNEL staining(Neurons/400-fold field of view, n = 4)

DISCUSSION

In the spinal cord, apelin has physiological and pathological functions[3]. In the present study, apelin was highly expressed in the cytoplasm and nerve fibers of anterior horn neurons of the normal spinal cord, consistent with previous results that high apelin expression was detected in the human spinal cord[4]. This indicates that apelin may be synthesized by the endoplasmic reticulum of neurons. After acute SCI, APJ receptor gene expression has been shown to be enhanced[5-9], leading to increased apelin synthesis from neurons. This result is agreement with our observation that apelin expression significantly increased 2 hours post injury. Ischemia or reperfusion-induced secondary SCI further induces neuronal death by necrosis and apoptosis[10-12], and subsequently reduces the number of neurons that can synthesize or secrete apelin, resulting in a reduction in apelin expression. Therefore, during 6-72 hours post SCI, apelin expression gradually decreased, which is consistent with cell death post SCI[7].

Apelin was highly expressed in nerve fibers but not expressed in neural cells following SNL. Spinal cord neuronal apoptosis may correlate with an increase in apelin expression that is induced by APJ receptor gene expression enhancement after stress[13-15]. In addition,neural cells can release apelin via exocytosis. As the endogenous ligand of GPCR, apelin binds to GPCR on the surface of neural cells and induces a series of biological effects in cells[16-19], which plays a role in nerve repair.In vitro cultures of cortical neurons maintained in serum-free media exhibited signs of apoptosis with degradation of nuclei after 24 hours[8]. Interestingly, apelin-13(1.0-5.0 nm) significantly inhibited cell death[20-22]. Apelin-13 can reduce active oxygen production in serum,mitochondrial depolarization, cytochrome C release and caspase-3 activation[23-25]. Apelin is an endogenous adipocyte factor that has neuroprotective effects. It can inhibit apoptosis and toxicity-induced cell death through cellular and molecular mechanisms[26-28]. In this study,the number of apoptotic neurons and glial cells was significantly reduced following apelin protein injection compared with the control group, while the peak of apoptosis occurred earlier. These results demonstrate that apelin protein can protect spinal cord neurons and reduce motor neuron apoptosis in the anterior horn,consistent with previous results[8,29].

In conclusion, apelin protein reduces motor neuron apoptosis in the anterior horn and delays the onset of apoptosis. Thus, apelin may play a role in the functional recovery of the nervous system and may have potential use as a novel target to treat SCI[30-32].

MATERIALS AND METHODS

Design

A randomized, controlled, animal experiment.

Time and setting

The experiment was performed at the Laboratory of Third Xiangya Hospital, Central South University, China from February 2009 to December 2010.

Materials

Adult Sprague-Dawley rats (n =119), regardless of gender, weighing 180-240 g, were provided by the Laboratory Animal Center of Central South University (No.SCXK (Hunan) 2006-0002). The experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals,issued by the Ministry of Science and Technology of the People’s Republic of China[33].

Methods

Animal grouping

Three experiments were performed. In experiment 1, 35 rats in total were used. Rats were randomly assigned to the normal (n = 5) and SCI (n = 30) groups. The SCI group was further subdivided into 2, 6, 12, 24, 48 and 72 hour groups (n = 5 per group). In experiment 2, 36 rats in total were used. Rats were assigned to the control (n = 6)and SNL (n = 30) groups. The SNL group was subdivided into 1, 3, 7, 14 and 21 day groups (n = 6 per group). In experiment 3, 48 rats in total were used. Rats were assigned to the control and apelin groups (n = 24). The apelin group was subdivided into 4, 8, 24 and 72 hours,and 7 and 14 days groups (n = 4 per group).

Experiment 1

Establishment of SCI model: The SCI model was established according to a previously published method[16].

Briefly, the spinal dura mater was exposed, and a cylindrical metal rod (2.0 mm in diameter, 11.2 cm in length and 10.24 g in weight) was dropped from a height of 5 cm under the guidance of a thin plastic tube to induce SCI. Successful injury was established if rats exhibited tail spasm, bilateral hind limbs and body retraction and flutter, followed by bilateral hind limb paralysis after 1 minute.

Histological detection of injured spinal cord in SCI models: At each time point after model establishment, L4-6segments of spinal cord were harvested, fixed in 4% (w/v)paraformaldehyde, paraffin embedded and cut into serial sections at 5 μm thickness. Five sections were used at each time point, dewaxed, and subjected to routine immunohistochemical staining. Mouse anti-rat apelin (1:200; M-77, Santa Cruz Biotechnology Inc., Santa Cruz,CA, USA) was used as the primary antibody, and biotinylated goat anti-mouse IgG (Wuhan Boster, Wuhan,China) was used as the secondary antibody. The sections were visualized with diaminobenzidine (Wuhan Boster; AR1022) and the reaction was controlled under a light microscope (Olympus, Tokyo, Japan). Five sections from each animal were used. Three fields of view in a circle of 1-mm diameter around the injury site were selected from each section using the HPIAS-1000 color pathological image analysis system (LKB, Uppsala,Sweden) to measure absorbance of positive cells in the unit area.

Experiment 2

Establishment of SNL model: Rats were anesthetized by intraperitoneal injection of 2% (w/v) pentobarbital sodium(40 mg/kg), and the sciatic nerve of the right hind limb was exposed. One 4.0 thread, 1 cm below the piriformis muscle, was permeated through the nerve, and the sciatic nerve was ligated[7]. Rats were sacrificed at 1, 3, 7,14 and 21 days. The control group did not receive any treatment. The L4-6segments of the spinal cord were harvested for immunohistochemical staining using the same method described in experiment 1.

Experiment 3

Establishment of sciatic nerve disconnection and intervention: In the apelin group, the sciatic nerve was disconnected and sutured. Apelin protein (Santa Cruz Biotechnology) was injected into a silica gel tube. The disinfection and exposure procedures were performed according to experiment 2. The sciatic nerve, 0.5 cm from the infrapiriform foramen, was disconnected and the silica gel tube was sheathed at the distal end (1.0 mm inner diameter, 1.0 cm length), fixed and sutured. The two ends of the tube were fixed using geoline (supplementary Figure 10 online). The apelin and control groups were injected with 30 μL of apelin protein antigen and 0.9% (w/v) normal saline, respectively, using the silica gel tube after model establishment. Rats were reperfused for 4, 8, 24 and 72 hours, and 7 and 14 days. The L4-6segments of the spinal cord were harvested for immunohistochemical staining using the same method described in experiment 1.

Detection of apoptotic cells using TUNEL

Apoptosis was detected using TUNEL[14]. Sections were treated with TUNEL reaction solution (terminal deoxynucleotidyl transferase (TdT) enzyme and digoxin-labeled nucleotide mixture; Beijing Zhongshan, Beijing, China),incubated with alkaline phosphatase(AP-anti-DIG-Fragment) at 37°C for 30 minutes, colorized with NBT/BCIP for 5-15 minutes, stained with nuclear fast red, dehydrated and mounted with neutral gum.

A phosphate buffered saline wash was performed between each step. Four non-overlapping fields of view from each section were observed (×400) and apoptosis-positive cells were quantified.

Statistical analysis

Measurement data were expressed as the mean ± SD.

Multiple-sample comparison was performed using one-way analysis of variance of repeated measurements.Intergroup differences were compared using LSD-t test.

Data were analyzed using SPSS11.0 software (SPSS,Chicago, IL, USA). A value of P ＜ 0.05 was considered statistically significant.

Author contributions:Zhiyue Li conceived and designed this study, and wrote and revised the manuscript. Qun Zhao provided and integrated experimental data. Siyin Feng, Bin Yang and Suling Zhang conducted the experiments. Yong Zhuang contributed to the evaluation of the study. Weiguo Wang provided technical support.

Conflicts of interest:None declared.

Ethical approval:This study received permission from the Animal Care and Research Committee of Central South University, China.

Supplementary information:Supplementary data associated with this article can be found in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 20, 2011 after selecting the “NRR Current Issue” button on the page.

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