Expression of Slit and Robo during remodeling of corticospinal tract in cervical spinal cord in middle cerebral artery occlusion rats

Slits and Robos were associated with the generation of axons of corticospinal tract during the corticospinal tract (CST) remodeling after the cerebral ischemic stroke (CIS). However, little is known about the mechanism of CST remodeling. In this study, we detected the expression of Slits and Robos in middle cerebral artery occlusion (MCAO) rats to investigate the roles of Slits and Robos in the CIS. MCAO model was established using modified Zea Longa method. Beam walking test (BWT) was conducted to evaluate the motor function. The images of the track of cortical spinal cord beam on day 7, 14 and 21 were observed by anterograde CST tracing. Biopinylated dextan amine (BDA) was used to mark CST anterogradely. Expression of GAP-43 mRNA and GAP-43 protein in cervical spinal cord was detected by Real-Time PCR and Western blot analysis, respectively. The expression of Slit1, Slit2 and Robo1 in cervical spinal cord was detected by immunofluorescence staining. The scores in the model group were significantly reduced compared to sham-operation group on day 7 (P < 0.001), 14 (P < 0.001) and 21 (P < 0.001), respectively. There was no significant difference in the score on day 7, 14 and 21 of the sham-operation groups (P > 0.05). In contrast, significant increase was noticed in the scores in model group, presenting a time-dependent manner. More CST staining fibers could be observed at the degenerative side in the model group compared with that of the sham-operation group on day 21. GAP-43 mRNA expression in the model group showed significant increase compared to that of sham-operation group on day 14 (P = 0.015) and 21 days (P = 0.002). The expression of GAP-43 protein in model group showed significant increase compared to that of sham-operation group on day 14 (P = 0.022) and day 21 (P = 0.008), respectively. The expression of Slit1 and Slit2 showed increase on day 14 and day 21, while the expression of Robo1 showed significant decrease in MCAO rats. Up-regulation of Slit1 and Slit2 and the downregulation of Robo1 may be related to the axons of CST midline crossing in spinal cord of MCAO rat during the spontaneous recovery of impaired motor function.


Background
Cerebral ischemic stroke (CIS), one of the most serious diseases threatening the health of human beings [1,2], is defined as ischemic necrosis or cerebromalacia induced by disorder of cerebral blood or hypoxia accompanied by neurological function defect such as motor dysfunction. Remodeling of corticospinal tract (CST) is considered a key event for the recovery of motor function after CIS [3]. Under normal conditions, axons of CST were localized at the preliminary sites and were inhibited to pass through the midline of the spinal cord. Nevertheless, in the presence of CIS, axons of CST that stemmed from the intact cortex could pass through the midline of spinal cord, which involved in the reconstruction of neural network of the damaged sites after CIS.
Slits (i.e. Slit 1-3) and Robo receptors (i.e. Robo1-4) are crucial for the passing of axons through the midline of spinal cord that is closely related to the CST. Indeed, the Slit-Robo signaling had been confirmed to participate in the autocrine/ juxtaparacrine regulation of axon fasciculation. For example, co-expression of Slit and glypican-1 mRNA was found in the reactive astrocytes of the injured adult brain tissues [4]. In focal cerebral infarction rats, electroacupuncture intervention significantly improved the neurological function and obviously upregulated the expression of cerebral Slit 2 and Robo 1 proteins [5]. Moreover, expression of netrin-1, Slit-1 and Slit-3 but not of Slit-2 was detected in cerebellar and spinal cord lesions [6]. Nevertheless, the roles of Slit-Robo signaling pathway in the focal cerebral infarction are still not well defined.
GAP43, a nervous tissue specific protein, is highly expressed in neurons during development and nerve regeneration. As is known to all, GAP43 is implicated in neural axon outgrowth, long-term potentiation, signal transduction, and neurotransmitter release. In the presence of neural damages, GAP43 is highly expressed. This led us to investigate the roles of GAP43 in the pathogenesis of CIS.
In this study, we aimed to investigate the expression of Slit1, Slit2 and Robo1 expression in the cervical cord midline in the rats with focal cerebral infarction. Specifically, we detected the expression of Slit1, Slit2 and Robo1 during the process of CST remodeling in cervical spinal cord in MCAO rats.

Animals
Forty-eight adult male Sprague Dawley (SD) rats (250-300 g; certificate No.: SCXK Lu 2014-0007) were provided by Lukang Pharmacy (Linyi, China). The rats were kept in a controlled environment at a constant temperature of 25 ± 1 °C and a humidity of 50% ± 10%. All the animals were free access to food and water on a 12 h/12 h light/dark cycle for at least 21 days under standard conditions before any treatment.

MCAO construction
MCAO was established according to Zea-Longa's method, with slight modifications [7]. In brief, the rats were intraperitoneally anesthetized with 4% chloral hydrate (0.7 ml/100 g). Then the anterior cervical tissues were cut longitudinally to expose the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). A small incision was made at a position that was about 4 mm from the CCA branch, and then a line was inserted into the blood vessel at a depth of 18 mm.

Experimental design
The rats were randomly divided into sham-operation group (n = 24) and the MCAO model group (n = 24), according to the random number table method. The rats in the sham-operation group were given a ligation of right carotid artery without inserting the line. In the model group, MCAO induction was conducted on day 1. BDA injection was performed in both groups on day 7, together with behavioral tests on day 7, 14 and 21, respectively. Animals were sacrificed on day 7, 14 and 21, followed by sample collection for the subsequent tests, including immunohistochemistry, Western blot analysis, and Real-Time PCR.

Neurobehavioral evaluation
Behavioral testing was performed by experienced investigators blinded to the experimental groups. The performance of animals in behavioral tests was assessed during the light portion of the light-dark cycle. The methods for each behavioral test are listed as follows.

The beam walking test
To evaluate the motor function of rats, the beam walking test (BWT) was conducted on day 7, 14 and 21 according to the previous description [8]. Briefly, rats were placed on a beam with a size of 122 cm × 2.5 cm × 75.5 cm, and were trained to walk along the beam to reach the opposite side of the beam for three trials about 3 days before the experiment [9]. A 0-7-point scale modified from Goldstein method [10] was utilized to evaluate the locomotor function of the animals.

The modified grip-traction test
The rats were required to grasp a plastic tube placed in an horizontal direction (0.6 cm in diameter) with their forward claws. The tube was about 45 cm above a desk. Then we determined the muscle strength and recorded the time for falling.

The rotarod test
Three days before MCAO induction, the rats were trained on a rotated bar (4-35 rotation per min). All the animals were trained for 3 days, with a frequency of three times per day lasting for 5 min, respectively. Then we recorded the time of animals with no falling when walking on the rotated bar. The time interval for the test was 15 min. The averaged value was obtained after two tests.

Anterograde corticospinal tract tracing
Biotinylated dextan amine (BDA, 0.2 µl) was injected to 4 sites. For the selection of injection sites, two sites were fixed in the position that was about 1 mm and 2 mm from the anterior and posterior bregma, while two sites were fixed in the position that was about 3.5 mm and 4 mm to the lateral bregma. After deep anesthesia, the rats were positioned on a stereotaxic apparatus via a finely drawn glass capillary on day 7. For each site, the BDA was injected at a depth of 1.5 mm, and the needles were dwelled for 2 min after injection.
Two weeks after BDA injection, rats were anesthetized and sacrificed for the subsequent analysis. Cervical spinal cord tissues obtained from C4 to C6 segments were fixed by 4% paraformaldehyde overnight, followed by dehydration in sucrose with a concentration of 10%, 20% and 30%, respectively. The tissues were then embedded by paraffin, and the sections (7 µm) were placed in 0.5% H 2 O 2 for 15 min, followed by washing three times with tris-buffered saline (TBS) solution. Subsequently, the sections were incubated in TBS containing 0.3% Trition X-100 at 4 °C for 6 h, and then were incubated overnight with HRP. The sections were presented in DAB solution for 15 min and were attached to the antistripping slide. Finally, the images were observed by light microscope.

Quantitative real-time PCR
Total RNA was extracted from the tissues of the left cervical spinal cords using TRIzol reagent (Thermo Fisher Scientific, CA, USA). The cDNA synthesis was carried out using the Transcriptor First-Strand cDNA Synthesis kit (Roche Diagnostics, Basel, Switzerland). Quantitative PCR was performed using 2 × SYBR Green qPCR Mix (Aid lab Biotech, Beijing, China). PCR amplification was performed using a Real-Time PCR System (Agilent Tech, CA, USA) using the following specific primers: β-actin, 5′-GCC TTC CTT CCT GGG TAT GG-3′, 5′-ACG CAG CTC AGT AAC AGT CC-3′; GAP-43, 5′-ACC ACT GAT AAC TCG CCG TC-3′, 5′-CTA CAG CTT CTT TCT CCT CCTC-3′. The amplification conditions were as follows: denaturation at 94 °C for 5 min, 42 cycles at 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s. The amplification results were evaluated using the ΔΔCq method as previously described [11].

Western blot analysis
Protein was extracted from the left cervical spinal cord homogenized in RIPA lysis buffer. The protein concentration was evaluated based on BCA method, followed by separation by 10% SDS-PAGE gels. Afterwards, proteins were transferred onto polyvinylidene difluoride membranes under 80 V for 30 min and 100 V for 2 h. The membranes were blocked with 5% non-fat dry milk for 1 h and incubated at 4 °C overnight with primary rabbit anti-GAP-43 (1:20,000, Abcam, UK). Then the membranes were incubated with secondary antibody, goat anti-rabbit IgA (1:3000, Bioss, Beijing, China), for 1 h at room temperature. The beta-actin served as the internal standard. Finally, the band gray values were analyzed via the application of Bio1D software.

Immunofluorescence
Cervical spinal cord tissue sections (6 μm) were soaked in acetone for 15 min and were incubated with 10% goat serum (0.01 mmol/L) at room temperature for 1 h. The slices were incubated with primary antibody overnight at 4 °C, including mouse anti-Slit1 (1:100, Abcam), rabbit anti-Slit2 (1:100, Abcam), and rabbit anti-Robo1 (1:10, Abcam). Upon washing with PBS, the slices were incubated with secondary antibodies at room temperature for 2 h, including Alexa Fluor488-conjugated goat anti-rabbit IgG (1:400; H&L, ab150077), Alexa Fluor 555-conjugated goat anti-mouse IgG (1:400; H&L, ab150114). DAPI solution (1:500) was added to the slices. After fluorescence quenching, the images were observed under a fluorescence microscope (ZEISS 780). SPSS 18.0 (SPSS, Chicago, USA) was utilized for the data analysis. All the data were expressed as mean ± standard error of mean. Student's t-test or the Mann-Whitney test was used for the comparison of measurement data between the two groups. P < 0.05 was statistically significant.

Neurological functional outcome and lesion volume
For the neurological function evaluation, the scores obtained based on Goldstein method in the model group showed significant decline compared to that of sham-operation group on day 7 (P < 0.001), day 14 (P < 0.001), and day 21 (P < 0.001), respectively (Fig. 1a, paired sample t-test). No significant differences were noticed in the scores on day 7, 14 and 21 in the sham-operation group (P > 0.05, Fig. 1a). By contrast, significant increase was noticed in the scores in model group, presenting a time-dependent manner. Compared with sham control, the grip-traction time on day 7, 14 and 21 showed significant decline in the MCAO group (P < 0.05, Fig. 1b). The residual time on rod in the MCAO group was significantly shorter on day 7, 14 and 21 compared with the sham control (P < 0.05). With the time went on, the residual time on rod in the MCAO group showed significant increase on day 7, 14 and 21 (P < 0.05, Fig. 1c). TTC staining showed that the infarction size showed significant decrease on day 14 and day 21 compared with that on day 7 (P < 0.05, Fig. 1d, e). This implied that motor function in MCAO rats showed significant reduction after surgery compared to that of sham-operation group. Nevertheless, these tests indicated that the motor function of MCAO rats could spontaneously recovered to some extent.

Midline-crossing CST axon sprouted into the denervated side of the cervical cord
About 14 days after injection, BDA was traced at CST of cervical spinal cord in sham-operation group and model group, respectively (Fig. 2a, c). In addition, more fibers stained in the CST region were available in the model group compared with the sham-operation group on day 21 (Fig. 2b,   Fig. 1 Motor function and cerebral infarction size at the corresponding time point. The motor function was determined using beam walking test (a), the modified grip-traction test (b) and rotarod test (c). The infarction size was evaluated using the TTC staining. Infarc-tion size in each group (d). Infarction rate in each group (e). Data were represented as mean ± standard error of mean (SEM). n = 24; *P < 0.05 vs. model group d). This implied that axons of CST could pass through the midline and entered the denervated side on day 21.

Expression of GAP-43 in the cervical spinal cord
In the model group, expression of GAP-43 mRNA showed significant increase compared to that of sham-operation group on day 14 (P = 0.015) and day 21 (P = 0.002), respectively (Fig. 3a). Similarly, expression of GAP-43 protein in model group showed significant up-regulation compared to that of sham-operation group on day 14 (P = 0.022) and day 21 (P = 0.008) (Fig. 3b, c). This implied that the CST axons in the denervated side remodeling on day 14, which lasted until day 21.

Immunohistochemical staining of Slit1, Slit2 and Robo1 in the cervical spinal cord
The protein expression of Robo1 was significantly downregulated as revealed by immunohistochemical staining. Meanwhile, the expression of Slit1 and Slit2 protein was significantly up regulated in the model group on day 14 and day 21 (Figs. 4,5,6). By contrast, compared with the shamoperation group, there was no significant differences for the expression of Slit1, Slit2 and Robo1 on day 7 in the model group (P > 0.05). On this basis, Slit1, Slit2 and Robo1 were involved in the process of CST remodeling after the surgery.

Discussion
CIS is the most common cause for long-term disability in adults [12,13], and most of the patients (approximately 75%) show various motor dysfunction [14]. According to the previous description, there was CST axonal loss in MCAO rats [15]. CST is a beam linked the sensorimotor cortex and the ventral horn motor neuron in spinal cord, which dominates the voluntary movement of skeletal muscle. In cases of CIS, there would be loss of domination of cortex for the contralateral motor neuron, which subsequently led to deficiency of contralateral motor function and a spastic paralysis. In a previous study, Murphy and Corbett reported spontaneous improvement of motor function in MCAO rats after CIS surgery [16]. Meanwhile, CST could pass through the midline of spinal cord to form new synapses of motor unit in the denervated side to promote the recovery of motor function in animal models with cerebral injury [17]. Therefore, the remodeling of CST axons promoted the CST midline passing through the denervated side, which was vital for spontaneous motor function recovery in CIS patients.
GAP-43 has been well acknowledged as a marker for axon growth. As a unique membrane-associated protein, it played crucial roles in the development and remodeling of the neurons. GAP-43 protein used for the labeling of axonal growth is mainly expressed at the regenerated axonal terminal [18][19][20]. Previously, up-regulation of GAP-43 was associated with the motor function recovery in both brain and spinal cord injury animal models [21][22][23]. In our study, the motor function recovery can be observed in the model group. In addition, the expression of both GAP-43 mRNA and GAP-43 protein in the model group showed significant increase after surgery on day 14 and day 21 compared with the sham-operation group, demonstrating the presence of the CST remodeling in MCAO rats at the denervated sides. BDA stained CST axons were found at the denervated side on day 21 after modeling, which showed that the CST axons presented at the denervated side were derived from the innervated side. This process mainly occurred on day 21 after surgery.
Nowadays, extensive studies have focused on the roles of Slit1, Slit2 and Robo1 in the nervous system. For instance, Slit1 was merely expressed in nervous system, while Slit2 was expressed mainly in nerve tissues. Robo1 was expressed in the central nervous system of mammalians and was uniformly distributed in axon. Besides, Robo1 could bind to all Slits proteins [24], which could be expressed by motor neurons [25,26]. Our finding suggested that the up-regulation of Slits and down-regulation of Robo may activate the CST midline crossing upon the sprouting of the axons at the innervated side of spinal cord in MCAO rats. This indicated that up-regulation of Slits and/or down-regulation of Robos might prompt motor recovery after CIS.
In the process of CST remodeling, the midline repulsion contains two courses: guiding the axons to cross the midline; and preventing the axons from re-crossing the midline once they crossed or under normal conditions. The Slit-Robo signaling pathway seemed to be activated in both of these processes. Slit-Robo pathway, acting as a signaling of repulsion, was imperative for maintaining the ipsilateral axon pathways under normal conditions [27][28][29]. In a previous study, Slit-Robo pathway played a key role in guiding dorsally projecting cranial motoneurons and facilitating the exit of neural tube [30]. Robo1 receptor could respond to Slits in spinal motor axons, which thereby repelling axons in vivo and in vitro [25,31,32]. Such repulsive reaction might be related to a self-silencing mechanism [33]. Suppressing the expression of Robo1 can promote axon outgrowth and midline crossing, while over-expression of Robo1 repelled the axons from the midline and prevented their re-crossing [34][35][36]. Similarly, our data showed that Robo1 was downregulated in MCAO rats in the presence of axons at the denevated side of cervical spinal cord.
To our best knowledge, Slit1, Slit2 and Robo1 could regulate the CST remodeling process in the spinal cord, however, there are still some disputes on the expression pattern. Liu [37] reported that the Slit2 expression began to raise about 7 days after spinal cord injury (SCI), and reached the peak level on day 14. However, the receptor Robo1 showed no significant changes at all time points, which indicated that Slit2-Robo1 signal pathway did not involve in the CST remodeling after SCI. In our study, the expression of Slit1 and Slit2 showed significant increase after surgery on day 7. In a previous study, Li et al. [38] indicated that 48 h following SCI in rats, up-regulation of Slit2 and down-regulation of Robo1 hindered the CST remodeling in spinal cord. Our findings were on the contrary, which might imply that Slits and Robos exerted different roles in different time point, and the animal model could also be a possible cause for it.
Indeed, there are some limitations in our study. We could not illustrate the exact mechanism of how Slit1, Slit2, Robo1, and GAP43 in the pathogenesis of CIS, despite we presented their changes in the MCAO rats. In future, more studies involving the silencing of Slit1, Slit2, Robo1, and GAP43 are required to further illustrate their exact roles in the CIS.

Conclusion
In summary, the CST remodeling occurred with the spontaneous motor function recovery from 14 to 21 days after MCAO. This was associated with the downregulation of Robo1 and up-regulation of Slit1 and Slit2.