Alterations in the spinal cord and ventral root after cerebral infarction in non-human primates

Abstract

Backgrounds:

Cerebral infarction does not only cause focal injury in the ischemic site, but also secondary non-ischemic damage at the remote areas of nervous system associated with the primary focus.

Objective:

This study investigated the changes in the spinal cord and ventral root after middle cerebral artery occlusion (MCAO) in cynomolgus monkeys (Macaca fascicularis).

Methods:

Adult male cynonolgus monkeys (4–5 years, 5.5–7.5 kg) were subjected to MCAO (n = 6) or sham surgery (n = 4). After 12 weeks, spinal cords and the ventral roots were harvested. Morphometric alterations in the spinal cord were detected at C5 and L5 levels via immunofluorescence. The profiles of C5 and L5 ventral roots were displayed by toluidine blue staining and transmission electron microscopic examination.

Results:

Significant axonal loss in the contralateral corticospinal tract and abnormally enlarged axons in the ipsilateral were observed in monkeys with MCAO. The number of neurons in the contralateral ventral horn got declined while that in the ipsilateral was almost unaffected after MCAO compared with sham controls. Glial activation post-MCAO was observed in the bilateral corticospinal tract and the ventral horn. Aberrant nerve fibers appeared frequently in the contralateral ventral roots of MCAO monkey but rarely in the ipsilateral.

Conclusions:

These results indicate that focal cerebral infarction leads to pathological alterations in the spinal cord and ventral roots in non-human primates.

Keywords

Cerebral infarction secondary damage spinal cord ventral root non-human primates

1 Introduction

Secondary damage occurs at the remote non-ischemic areas of the nervous system connecting to the primary focus after cerebral infarction (J. Zhang et al., 2012). A great many clinical and experimental studies revealed the neuronal degeneration and glial activation in the thalamus, hippocampus or substantia nigra after middle cerebral artery infarction(Butler et al., 2002; Wang et al., 2012; Y. Zhang et al., 2011). Moreover, secondary damage after cerebral infarction is not only within the brain, but extending beyond the cranial region. In both human and rodents, Wallerian degeneration of the corticospinal tract (CST) was observed in the spinal cord level after cerebral infarction(Buss et al., 2005; Iizuka et al., 1989). Electromyography examinations displayed denervation changes in paralyzed muscles, such as the spontaneous potential, the reduction of motor unit number estimated (MUNE) or compound motor action potential (CMAP) in stroke patients and rodents, suggesting the secondary damage in the spinal motor neurons and/or peripheral nerves(Li et al., 2011; Lin et al., 2015; Lukacs, 2005). In our previous study, CST axonal loss, degradation of the ventral horn (VH) neurons and the ventral roots were seen in the cervical and lumbar enlargements of the spinal cord after middle cerebral artery occlusion (MCAO) in hypertensive rats (G. Dang et al., 2016).

As so far, basic researches on cerebral infarction were mainly conducted on rodents which are significantly different from human in neurological anatomy and pathophysiology. The differences have been frequently cited as potential reasons for barriers in the transformation of basic-science into clinical application. It is well-known, in neurological protection after stroke, that almost all effective neuroprotectants proved in rodents have not been confirmed in clinical trials (O’Collins et al., 2006). In addition, differences in secondary damage post-stroke between human and rodents were noticed as well. Even though CST fibers in the spinal cord lost gradually within several years after onset in stroke patients (Buss et al., 2005), our previous study showed the number of CST axons was reduced at the first week and maintained within 12 weeks post-MCAO in the contralateral spinal cord (G. Dang et al., 2016). Furthermore, beta-amyloid (Aβ) deposition was observed in the ipsilateral thalamus after MCAO in rodents (Hiltunen et al., 2009; Y. Zhang et al., 2011), while no correlation was shown between cerebrovascular diseases and thalamic Aβ deposition in human (Aho et al., 2006). Recently, a study conducted in marmoset found no Aβ deposition in the thalamus after MCAO (Lipsanen et al., 2013), suggesting that nonhuman primates (NHPs) might be more similar to human in the secondary damage after cerebral infarction than rodents. Based on our previous work conducted in rats, we further explored alterations in the spinal cord focusing on the CST and VH and ventral roots after MCAO in cynomolgus monkeys (Macaca fascicularis) here.

2 Materials and methods

2.1 Ethics approval

Animal use protocols were authorized by the Institutional Animal Ethical Committee of Sun Yat-Sen University and accredited by the Assessment and Accreditation of Laboratory Animal Care (AAALAC).

2.2 Animal preparation and MCAO model

Ten male cynomolgus monkeys (4–5 years, 5.5–7.5 kg, provided by Guangdong Landau Biotechnology Co., Ltd, Guangzhou, China) were used in this study. The animals were housed individually at a temperature of 24–28°C with a 12-hour light/12-hour dark cycle. All animals were fed twice daily and had ad libitum access to clean drinking water. The cynomolgus monkeys were assigned randomly to receive MCAO (n = 6) or sham operation (n = 4). The MCAO procedure has been reported in detail previously (Chen et al., 2015). Anesthesia was induced with ketamine (10 mg/kg, intramuscular injection) and then maintained with isoflurane (1.5–3%, inhalation). Temperature, blood pressure, heart rate and respiration were monitored. The monkeys were fixed in a stereotaxic frame and a pterional craniotomy was performed. Afterwards, the left middle cerebral artery (MCA) was exposed and occluded in the distal M1 branch of MCA by bipolar electrocoagulation. The occluded MCA was cut to avoid recanalization. Finally, the bone window was waxed and the incision was sutured closed. In sham-operated group, the MCA was only exposed but not be occluded or cut. Penicillin was administered to prevent infection within 3 days after operation (0.4 million IU, intramuscular injection) and the wounds were examined carefully for signs of infection. Intensive care was provided until animals were able to self-care, eat and drink. Neurological assessment was performed in all animals using a standardized stroke-related neurological deficit score (Kito et al., 2001) before and 1 week post-operation, and the complete description of the neurological function is available in Chen et al. (2015).

2.3 Tissue preparation

At 12 weeks after operation, all animals were deeply anesthetized by phenobarbitone (25 mg/kg) intramuscularly. After thoracotomy, monkeys were perfused with 2000 ml saline transcardially and followed by 3000 ml 4°C 4% paraformaldehyde. The cervical and lumbar enlargements of the spinal cord were collected and fixed in 4 °C 4% paraformaldehyde for 24 hours. After dehydrated in 20% and 30% sucrose sequentially, the spinal cords were cut in 10-μm coronal slices on a cryostat (Leica, CM1900, Germany) for immunofluorescence examination. The C5 and L5 ventral roots were removed immediately after laminectomy and fixed in the 4°C 4% paraformaldehyde and 2.5% glutaraldehyde buffer mixture fast. The ventral roots were sectioned crossly with an ultramicrotome (Leica, UC6, Germany) for toluidine blue staining (semi-thin, 750 nm) and transmission electron microscopic (TEM) examination (ultra-thin, 80 nm).

2.4 Immunofluorescence

A series of sections of the spinal cord were selected at intervals of every tenth sections. Briefly, sections were treated with citrate buffer (85 °C, 0.01 m/L, pH6.0) for 5 minutes followed permeation with 0.3% Triton for 30 min, then blocked with 5% normal goat serum for 1 h at room temperature. Subsequently, sections were incubated with the following primary antibodies at 4°C overnight respectively: mouse anti-NF-200 (a marker of axon, 1 : 800, Sigma-Aldrich Cat# N5389, RRID:AB_260781), mouse anti-NeuN (a marker of neuron, 1 : 500, Millipore Cat# MAB377, RRID:AB_2298772), rabbit anti-Iba-1 (a marker of microglia, 1 : 500, Wako Cat# 019-19741, RRID:AB_839504) or rabbit anti-GFAP (a marker of astrocyte, 1 : 500, Abcam Cat# ab48050, RRID:AB_941765). Negative control sections were incubated with 0.01PBS instead. Afterwards, sections were incubated with Alexa Fluor 555-conjuncted goat anti-mouse IgG (1 : 1000, Cell Signaling Technology Cat# 4408, RRID:AB_10694704) or Alexa Fluor 488-conjuncted goat anti-rabbit IgG (1 : 1000, Cell Signaling Technology Cat# 4412, RRID:AB_1904025) at room temperature for 1 hour. Finally, sections were mounted with Fluoroshield with DAPI (Sigma, #F6057) and examined under a fluorescence microscope (BX51; Olympus).

2.5 Toluidine blue staining and TEM examination

Semi-thin sections of the ventral roots were stained with 0.1% toluidine blue for 2 minutes, rinsed, air-dried and mounted, and then observed under a light microscope (BX51; Olympus). Ultra-thin slices stained in saturated uranyl acetate and lead citrate were photographed under TEM (FEI Tecnai G2 Spirit Twin).

2.6 Image analysis and quantification

Total NeuN-positive cell numbers in the VH were counted for quantitative analysis. The number of NF-positive axons, Iba-1-positive cells and the density of GFAP-positive signals were analyzed in 3 non-overlapping fields in the dorsolateral funiculus (the main CST zone) or the VH. G-ratio of the fibers in the ventral roots were analyzed in 6 non-overlapping fields.

2.7 Statistical analysis

All data were presented as means±SEM. Statistical analyses were conducted using one-way analyses of variance (ANOVA) followed by Bonferroni corrections for multiple comparisons. Differences were considered significant at p < 0.05.

3 Results

3.1 Axonal loss in the CST

The number of NF-positive axons was calculated to evaluate the axonal loss of the main CST. When intact axons with different sizes in sham-operated group distributed evenly (Fig. 1a), residual axons in the contralateral CST after MCAO displayed fragmentation and distinct increased spacing in between (Fig. 1c). At C5 level, the number of NF-positive axons in the contralateral CST of MCAO monkeys was significantly reduced compared to that in the ipsilateral and the sham-operated group (610±62 vs. 1189±140 or 1771±101 per field, all p < 0.05) (Fig. 1d). Similar pathological changes also occurred at L5 level, the number of NF-positive axons in the contralateral CST was also decreased after MCAO (747±87 per field vs. 1396±92 per field in the ipsilateral or 1911±140 per field in sham-operated group, all p < 0.05) (Fig. 1e). In the ipsilateral CST of MCAO monkeys, the number of NF-positive axons was less than that in the sham-operated group at both C5 and L5 levels as well (all p < 0.05) (Fig. 1d, e).

Fig. 1.

Expression of NF in the spinal cord. Immunoreactivity of NF in the CST of the cervical spinal cord at 12 weeks after sham operation or MCAO in cynomolgus monkeys (a–c). The number of NF-positive axons in C5 and L5 segments after sham operation or MCAO (d, e). Values are mean±SEM, n = 4 in sham-operated group and n = 6 in MCAO group, ^*p < 0.05, vs. the contralateral CST of sham operation, ^#p < 0.05, vs. the ipsilateral CST of MCAO monkeys. Ipsi. means ipsilateral, Contra. means contralateral. Scale bar = 50μm.

3.2 Enlargement of axons in the ipsilateral CST

In addition to the quantitative change, the cross-sectional area of individual NF-positive axon in the CST was altered after MCAO (Fig. 1b). In sham-operated group, the cross-sectional area of the axon arranges from 0.1 to 268μm², while there were about 86.95% axons with area of 0.1–20μm², 12.67% axons of 21–100μm² and 0.38% axons of 101–268μm² (Fig. 2). In the ipsilateral CST of MCAO monkeys, the maximum axon cross-sectional area was up to 521μm², and the percentage of large axons with area > 100μm² was increased in both C5 and L5 segments compared with the sham controls, while the percentage of axons with area < 100μm² was decreased at C5 level (Table 1) (all p < 0.05). Because the vast majority of axons in the contralateral CST were fragmented after MCAO, their cross-sectional areas were not analyzed here.

Fig. 2.

Frequency histograms of the NF-positive axon cross-sectional areas in the spinal CST of cynomolgus monkeys after sham operation.

Table 1

Comparison of the frequency distribution of the NF-positive axon cross-sectional areas in the ipsilateral CST. Values are mean±SEM, n = 4 in sham-operated group and n = 6 in MCAO group, ^* p < 0.05, vs. the sham-operated group

Area (μm2)	Percentage (%)	C5		L5
		Sham	MCAO	Sham	MCAO
0–20		85.20±1.21	71.90±4.18^*	82.86±6.51	84.86±2.07
21–100		14.00±1.35	20.40±2.42^*	16.93±6.48	14.20±1.85
101–280		0.83±0.16	7.61±1.96^*	0.22±0.04	0.88±0.32^*
>280		0	0.09±0.03^*	0	0.07±0.02^*

3.3 Neuronal degradation in the VH

NeuN-positive neurons remained contact in the VH of both the cervical and lumbar spinal cord after MCAO, whereas the immunoreactivity of NeuN was weaker than the sham-operated group (Fig. 3a–c). Moreover, the number of NeuN-positive neurons in the contralateral VH of infarction was less than that in the sham-operated group at C5 level (93±3.5 vs. 118.1±4.1 per section, p < 0.05) (Fig. 3d). At L5 level, the number in the contralateral VH of MCAO monkeys was decreased compared to the ipsilateral or the sham operation group (87.2±3.6 vs. 106.9±4 or 110.5±5.7 per section, all p < 0.05) (Fig. 3e). No significant difference was found between the number of NeuN-positive neurons in the ipsilateral VH of infarction and sham-operated group at both C5 and L5 levels.

Fig. 3.

Expression of NeuN in the spinal cord. Immunoreactivity of NeuN in the VH of the cervical spinal cord at 12 weeks after sham operation or MCAO in cynomolgus monkeys (a–c). The number of NeuN-positive neurons in C5 and L5 segments after sham operation or MCAO (d, e). Values are mean±SEM, n = 4 in sham-operated group and n = 6 in MCAO group, ^*p < 0.05, vs. the contralateral VH of sham operation, ^#p < 0.05, vs. the ipsilateral ventral horn of MCAO monkeys. Ipsi. means ipsilateral, Contra. means contralateral. Scale bar = 100μm.

3.4 Microglia activation in the CST and VH

In sham-operated group, Iba-1-positive microglia presented ramified with small soma and thin processes in both the CST and VH (Fig. 4a, d). After MCAO, most microglia displayed amoeboid in the CST (Fig. 4c) and aggregation of microglia with enlarged cell body and thick processes was observed in the VH (Fig. 4f) of the contralateral spinal cord. Furthermore, there were many ramified microglia showed larger soma and thicker branches in the ipsilateral CST of MCAO monkeys (Fig. 4b). Quantitative analysis showed that the number of Iba-1-positive microglia in the contralateral CST post-MCAO was greater than that in the ipsilateral and the sham-operated group at C5 level (21.7±2.2 vs. 11.2±0.8 or 3.4±0.4 per field, all p < 0.05) (Fig. 4 g), so did that in the VH (10.2±0.6 vs. 6.6±0.4 or 3.9±0.2 per field, all p < 0.05) (Fig. 4i). At L5 level, the number in the contralateral CST of MCAO monkeys and VH was also increased compared to that in the ipsilateral and sham-operated group (CST: 25.3±2.7 vs. 10.2±0.9 or 3.3±0.4 per field, VH: 12.3±0.8 vs. 8.3±0.5 or 4.2±0.2 per field, all p < 0.05) (Fig. 4h, j). Moreover, there were significant differences between the number of microglia in the ipsilateral spinal cord of the infarction and sham-operated group at both C5 and L5 levels (all p < 0.05) (Fig. 4g–j).

Fig. 4.

Expression of Iba-1 in the spinal cord. Immunoreactivity of Iba-1 in the CST (a–c) and VH (d–f) of the cervical spinal cord at 12 weeks after sham operation or MCAO in cynomolgus monkeys. The number of Iba-1-positive microglia in C5 (g, i) and L5 (h, j) segments after sham operation or MCAO. Values are mean±SEM, n = 4 in sham-operated group and n = 6 in MCAO group, ^*p < 0.05, vs. the contralateral side of sham operation, ^#p < 0.05, vs. the ipsilateral side of MCAO monkeys. Ipsi. means ipsilateral, Contra. means contralateral. Scale bar = 100μm.

3.5 Astrocytic scar in the CST and VH

GFAP staining displayed the soma and processes of astrocyte. In sham-operated animals, the astrocytic network in the CST and VH was relatively sparse, while an obvious astrocytic reaction was displayed in the spinal cord of MCAO monkeys (Fig. 5a–f). At C5 level, the density of GFAP-positive signal in the contralateral CST was increased to 171494±12647 pixels per field post-MCAO, which was greater than that in the ipsilateral or the sham-operated group (158657±9931 or 63871±4677 pixels per field, all p < 0.05) (Fig. 5g), so did that in the VH (174534±8552 vs. 131316±8098 or 96749±5765 pixels per field, all p < 0.05) (Fig. 5i). At L5 level, spinal astrocytic activation was also obvious after MCAO, and the density of GFAP-positive signal in the contralateral CST and VH was upregulated to 166939±17445 pixels per field and 182875±14546 pixels per field respectively, which were greater than that in the ipsilateral (121827±11675 pixels per field in the CST, 174241±12918 pixels per field in the VH) and the sham-operated group (76179±10034 pixels per field in the CST, 61786±3198 pixels per field in the VH) (all p < 0.05) (Fig. 5h, j). Significant differences between the density of astrocyte in the ipsilateral spinal cord of the infarction and the sham-operated group were observed at both C5 and L5 levels as well (all p < 0.05) (Fig. 5g–j).

Fig. 5.

Expression of GFAP in the spinal cord. Immunoreactivity of GFAP in the CST (a–c) and VH (d–f) of the cervical spinal cord at 12 weeks after sham operation or MCAO in cynomolgus monkeys. The density of GFAP-positive signal (astrocytic soma and processes) in C5 (g, i) and L5 (h, j) segments after sham operation or MCAO. Values are mean±SEM, n = 4 in sham-operated group and n = 6 in MCAO group, ^*p < 0.05, vs. the contralateral side of sham operation, ^#p < 0.05 vs. the ipsilateral side of MCAO monkeys. Ipsi. means ipsilateral, Contra. means contralateral. Scale bar = 50μm.

3.6 Degeneration in the ventral roots

Ventral roots were regular in sham controls showed by toluidine blue staining (Fig. 6a). After MCAO, many atrophic axons with thick myelin sheath were observed in the contralateral ventral roots (Fig. 6c) and a few abnormal fibers were seen in the ipsilateral (Fig. 6b). TEM examination showed the atrophic axon with thick myelin sheath (Fig. 6e) or axoplasmic accumulation of dystrophic membranous debris (Fig. 6f) post-MCAO.

Fig. 6.

Toluidine blue staining displayed no or few abnormal nerve fibers was seen in sham controls (a) or the ipsilateral C5 ventral roots (b), while abundant abnormal fibers with atrophic axon and thick myelin sheath (arrow) in the contralateral (c) were seen at 12-week after MCAO in cynomolgus monkeys. Normal fibers (d), atrophic axon with thick myelin sheath (e) and axoplasmic accumulation of dystrophic membranous debris (f) observed under TEM. Scale bar (in a–c) = 20μm, Scale bar (in d–f) = 1 nm.

4 Discussion

This study revealed axonal loss and enlargement, neuronal loss and glial activation in the spinal cords and aberrant fibers in the ventral roots at 12-week after MCAO in cynomolgus monkeys, suggesting the secondary alteration in the extracranial regions of the nervous system post-stroke in NHPs.

In the early phase of Wallerian degeneration, cytoskeletal protein of axon such as neurofilaments and microtubules is rapidly degraded to amorphous debris. The current study revealed axonal fragmentation and tremendous axonal loss in the contralateral CST of the spinal cord at 12 weeks after MCAO in cynomolgus monkeys. Wallerian degeneration of the CST in stroke patients has been investigated widely. Several studies showed degenerated intracranial CST within 3 months after onset in stroke patients by diffusion tensor imaging (DTI) or pathological examination(Buss et al., 2004; Matsusue et al., 2007; Puig et al., 2010; Thomalla et al., 2004). Moreover, postmortem examination displayed degenerating CST with irregular axon and myelin in the cervical spinal cord at 2 weeks after cerebral infarction, and clear pathological alteration of axons with increased spacing could be identified as far caudal as lumbar region by 5 weeks and hardly any axon could be detected after 1 year, while the amount of myelin rings reduced at 5 weeks post-stroke in patients (Buss et al., 2004; Buss et al., 2005). While spinal CST degenerated gradually in stroke patients, degradation and loss of CST fibers in the cervical and lumbar spinal cord of rats occurred at 1 week and did not deteriorate within 12 weeks post-MCAO (G. Dang et al., 2016; Iizuka et al., 1989). Although with highly similar morphologic alterations, Wallerian degeneration is greatly delayed and continues slowly over a number of years in humans compared to experimental studies in rats. Distinct scale and fiber caliber in the spinal cord might be the underlying reasons for the different timing of the secondary degeneration of the CST post-MCAO in human and rodents (Vargas et al., 2007). NHPs were more similar to human in neurological anatomy and physiology, which might make them better subjects for studying degeneration of the CST after stroke. This study revealed the CST axonal loss in the cervical and lumbar spinal cord in NHPs after MCAO, enriching evidences of Wallerian degeneration in the spinal CST post-stroke, laying the foundation for future researches on the dynamic secondary changes after stroke, and indicating an excellent animal model for studying the mechanisms of stroke-induced degeneration of CST.

In stroke patients and rodents, electrophysiological examination revealed spontaneous potential, MUNE reduction and CMAP decrease in the affected muscles, suggesting the secondary damage in the lower motor neurons and/or their axons (Li et al., 2011; Lin et al., 2015; Lukacs, 2005). The present results displayed neuronal loss in the contralateral VH of cervical and lumbar spinal cord at 12 weeks after MCAO in cynomolgus monkeys. In a postmortem study, Terao, et al. examined the cell populations, diameter and size distribution pattern in the VH in 4 unilateral stroke patients survived for 1–8 years, and found no significant difference between the contralateral and the ipsilateral VH or the controls (Terao et al., 1997). Nevertheless, Qiu, et al. reported that the cell number didn’t change, but the total VH area and the VH cell area were significantly decreased in the contralateral side in 45 patients suffered unilateral stroke for more than 6 months (Qiu et al., 1991). In these two postmortem studies (Qiu et al., 1991; Terao et al., 1997), subjects suffered ischemic or hemorrhagic stroke with variable location of the primary lesion, while all MCAO monkeys in this study had consistent infarct in unilateral cortex. Besides, both postmortem studies used Kluver-Barrera’s method for displaying neurons, whereas we here used immunofluorescence technique. Those might be the underlying reasons for the difference in quantification of VH neurons between human and monkeys. Still, the decrease of the VH area and VH cell area in human reported by Qiu, et al. suggested the secondary damage in the spinal cord after stroke. In MCAO rats, neurons in the VH surrounded by reactive microglia and neuronal loss in the contralateral VH was showed post-stroke (G. Dang et al., 2016; Wu et al., 1998). Degeneration of the ventral roots in cynomolgus monkeys further supported the damage in the lower motor neurons after cerebral infarction. Denervation might be one reason of the damage in the grey matter of spinal cord post-stroke, as well as the glial activation (Block et al., 2005; Wu et al., 1998).

In the central nervous system, microglia play as sentinels to survey and scan the microenvironment. Once stimulated by a threat, microglia rapidly become activated and transform from ramified to amoeboid phenotype which could eliminate cell debris and release bioactive molecules (Guruswamy et al., 2017). Several steps and intermediate stages can be identified during the transformation, including characteristics of process withdrawal, soma enlargement, hypertrophic cell with pseudopodia, etc. (Kettenmann et al., 2011). Secondary microglia reaction after middle cerebral artery infarction was found in the ipsilateral thalamus, hippocampus or substantia nigra (Block et al., 2005; Pappata et al., 2000). The current study showed microglia activation in the CST and VH of the spinal cord in cynomolgus monkeys at 12 weeks after MCAO. Both human and rats had microglia activation in the spinal CST after cerebral infarction at the chronic stage. In terms of the VH, postmortem study showed weaker microglia expression at 1–4 months than 4–14 days in stroke patients, and experimental study suggested that microglia was activated significantly in the first week with gradual decrease within 12 weeks after MCAO in rats correspondingly (G. Dang et al., 2016; Schmitt et al., 1998). In addition, most microglia in the contralateral CST of MCAO monkeys appeared amoeboid, which were clearing the debris of CST very possibly, and microglia in the contralateral VH were likely to be in an intermediate stage but their roles remain unknown. In recent years, microglia and macrophage are considered becoming polarized towards classically activated (M1) or alternatively activated (M2) phenotypes at various stages after CNS injury. While M1 phenotype releases proinflammatory mediators expanding tissue damage and impeding neuronal repair, M2 microglia and macrophage promote recovery by clearing cellular debris and releasing protective factors (Hu et al., 2015). Dynamic observation should be conducted to determine whether microglia in the VH of the spinal cord increased at the acute phase post-stroke and decreased later in NHPs, as well as the roles microglia played in different stages.

Astrocytes are the most abundant glial cells in the central nervous system. Glial scar formed by activated astrocytes is an important reason for hindering neurological recovery after neuronal injury (Pekny et al., 2014; Yiu et al., 2006). This study found obvious astrocytic proliferation in the CST and VH of the spinal cord of cynomolgus monkeys at 12 weeks after MCAO. In stroke patients, pathological changes in the astrocytic pattern were not found in the spinal cord within 2 weeks after onset (Buss et al., 2004), but moderate increase of astrocyte in the grey matter was observed 4 months later (Schmitt et al., 1998). In rats, astrocyte was activated in the spinal cord at 1 week after MCAO, which was gradually increased within 12 weeks and negatively correlated with neurological function recovery (G. Dang et al., 2016). It seems like astrocytic activation in the spinal cord post-stroke differs between species.

The ipsilateral spinal cord is also influenced after stroke partly due to the anatomical characters of the CST. In NHPs, there are 87% of CST fibers decussate in the medullary pyramids and descend through the contralateral dorsolateral tract of spinal cord, 11% fibers uncrossed descend on the ipsilateral dorsolateral tract and 2% on the ipsilateral ventromedial CST (Rosenzweig et al., 2009). Correspondingly, we noticed axonal loss of CST and glial reaction in the ipsilateral spinal cord after MCAO in cynomolgus monkeys in this study. In human, there are about 85–92% CST fibers crossed at the caudal of medulla oblongata descending on the contralateral dorsolateral funiculus of the spinal cord, while 8–15% uncrossed fibers descending on the ipsilateral anterior funiculus (Rea, 2015). Clinically, motor deficits including reduction of muscle force and dexterity were observed in the ipsilateral limbs in patients with unilateral stroke (Baskett et al., 1996; Noskin et al., 2008; Sunderland et al., 1999). Electromyography examination also showed decreased CMAP and increased jitter in the non-paralyzed limbs (Hunkar et al., 2012; Paoloni et al., 2010; van Kuijk et al., 2007). Although overuse of the non-paretic limbs after stroke may result in the electrophysiological alterations (Sato et al., 1999), transsynaptic change of the spinal neurons secondary to stroke cannot be neglected (Lukacs et al., 2009). Postmortem examination displayed degeneration of the ipsilateral anterior CST and glial activation in the ipsilateral grey matter of spinal cord after cerebral infarction (Buss et al., 2004; Schmitt et al., 1998). In rats, 96–98% of CST projections decussate in the medullary pyramids traveling at the contralateral lateral funiculus mainly and 2–4% undecussated fibers run in the ipsilateral dorsal and ventral funiculi. (Liang et al., 1991; Rouiller et al., 1991). Glial reaction was observed in the ipsilateral dorsal funiculus and VH in MCAO rats as well (G. Dang et al., 2016). However, no significant neuronal loss in the ipsilateral VH was observed in human, cynomolgous monkeys and rats after cerebral infarction.

More interestingly, we noticed the abnormal enlarged NF-positive axons in the ipsilateral CST of spinal cord in MCAO monkeys in this study (Fig. 1, Table 1). It is not clear the enlargement of axons indicating swelling (swollen axon as Buss et al. mentioned (Buss et al., 2004)) or growth. If they were the swollen axons, it was suggested that nerve fibers in the ipsilateral CST underwent different Wallerian degeneration phase from the contralateral ones at 12 weeks after MCAO in cynomolgus monkeys. However, if they were fibers growing, that might be an indication of compensation from the contralateral hemisphere. In our previous study, the grey matter volume in the contralateral supplementary motor area (SMA) was increased after cerebral infarction in patients with unilateral stroke (C. Dang et al., 2013). Anatomically, some axons derived from SMA project to the spinal cord via the ipsilateral CST in primates (Lacroix et al., 2004). It is presumed a potential correlation existed between the increased grey matter volume of the contralateral SMA and the enlarged axon in the ipsilateral CST of the spinal cord after stroke. Due to the significance of the contralateral hemisphere in neuroplasticity after stroke, secondary changes in the ipsilateral spinal cord after MCAO deserve more attention and the presumption mentioned above should be explored further.

Conflicts of Interest

The authors declare that they have no conflict of interest.

Footnotes

Acknowledgments

This study was supported by the National Key R&D Program of China (grant numbers 2017YFC1307500), the Natural Science Foundation of China (grant numbers 81371277 and 81571107), the Special Funds of Public Interest Research and Capacity Building of Guangdong Province (grant number 2014B020212003), grants from the National Key Clinical Department, National Key Discipline, the Guangdong Provincial Key Laboratory for diagnosis and treatment of major neurological diseases (grant number 2014B030301035), the Southern China International Cooperation Base for Early Intervention and Functional Rehabilitation of Neurological Diseases (grant number 2015B050501003), and the Project of Guangzhou Science Technology and Innovation Commission (grant number 201604020010).

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