Introduction

Spinal cord injury (SCI) is a devastating medical problem that causes serious disabilityand paralysis. Approximately 40 million people worldwide experience SCI every year1. The primary injury is caused by traumatic spinal cord damage2. The secondary injury can destroy nearby neurons that were notdamaged during the primary injury3. After the initial damage of theblood vessels in a spinal cord region, secondary injury causes a fall in microvascularblood flow that leads to ischemia and hypoxia, which exacerbate the primary injury4. In previous studies, spinal cord blood flow was often measured byDoppler ultrasound5. However, Doppler ultrasound can only measureblood vessels of approximately 100 μm in diameter6,damage to regional microvascular blood flow proximal to lesion site remains poorlyunderstood. In addition, an increase in intracellular free [Ca2+] resultsin the activation of the calcium-activated protease calpain, which is involved inneuronal apoptosis7. However, the changes of calcium influx ininjured axons of living animal after SCI remains unclear. Furthermore, the role ofmicroglia in SCI has been controversial with both beneficial and destructiveeffects8. Microglia can phagocytose cellular debris after SCI.They also can infiltrate and accumulate at the injured epicenter and secreteproinflammatory cytokines, which may aggravate secondary SCI9.

To reduce secondary injury after SCI, clinical and experimental studies have beenconducted to block the development of these abnormalities. Ecto-domainphosphorylation10 and fluoxetine treatment11 have been reported as potential methods for functional recovery after SCI.Although the effects of these therapeutic regimens are compelling, their clinicalapplications are limited. After the first demonstration of the experimental efficacy ofhigh dose methylprednisolone (MP) in acute experimental SCI12, MPhas been widely used in clinical treatment for SCI patients13.However, recent retrospective cohort studies have demonstrated a lack of statisticaldifference between SCI patients treated with and without MP14. Theefficacy of MP in SCI treatment remains controversial.

In previous laboratory studies, axons were assessed by biotinylated dextran amine (BDA)tract tracing15 and the intracellular calcium concentration in theinjured spinal cord was measured using the techniques of La3+ blockageand atomic absorption spectroscopy16. For these in vitromethods, tissue must be extracted from the spinal cord. For these reasons, the earlydynamic changes of SCI and MP's effect governing secondary injury remainunclear. In the present study, we took advantage of two-photon microscopy and spinalcord implanted window, which are able to image axonal dieback in the living mouse spinalcord over multiple hours. We also performed in vivo imaging of the regionalmicrovascular blood flow and calcium influx into axons at the edge of lesion site17. These in vivo methods allowed us to further ourunderstanding of early dynamic changes, as well as MP's effect on axonaldamage, microvascular blood flow and calcium influx into axons after SCI.

Results

MP attenuated axonal damage and neuronal death

We used two-photon microscopy to image the axonal dieback in the living mousespinal cord and investigate the effect of MP treatment after hemisection SCI(Fig. 1). Our results showed that the axons in thesham group (n = 6) remained intact during all imaging sessions after surgery.The severed axons dieback from the initial lesion site over time afterhemisection injury (Fig. 2A). We first imaged the injuredaxons at 30 min post-injury and measured the axonal dieback distance from theinitial lesion site. The respective axonal average dieback distances from theinitial lesion site at 8 h, 24 h and 48 h were 197.95 ± 42.87μm, 258.72 ± 30.79 μm, 292.26 ±40.54 μm in the saline-treated SCI group (n = 6) and 101.29± 29.89 μm, 142.04 ± 43.75 μm,167.58 ± 42.41 μm in the MP-treated SCI group (n = 6),respectively (Fig. 2C). At each time point, thesaline-treated group exhibited a greater axonal dieback distance than theMP-treated group (P < 0.01 for all). To investigate the pathologicalchanges and MP's effect on deep tissue after SCI, we measured thenumber of neurons at the edge of lesion site 3 days post-injury (Fig. 2B). The number of neurons was 48.71 ± 7.26cells/mm2 in the saline-treated group (n = 6) and 80.21± 5.76 cells/mm2 in the MP-treated group (n = 6). Thenumber of neurons was greater in the MP group than in the saline group (Fig. 2D, P = 0.007).

Figure 1
figure 1

In vivo two-photon imaging of the mouse spinal cord.

(A) The customized spinal stabilization device with an implanted window. (B)The mouse with an implanted window. (C) The segment of the spinal cordexposed for two-photon imaging. (D) The schema showing the implanted windowon the exposed T12 spinal cord segment.

Figure 2
figure 2

MP attenuated axonal damage and neuronal death after SCI.

(A) In vivo two-photon imaging of axonal dieback after hemisection spinalcord injury. Representative images of axons in the sham group (n = 6) at 30min, 8 h, 24 h and 48 h post-surgery. Representative images of axonaldieback in the saline-treated SCI group and the MP-treated SCI group at 30min, 8 h, 24 h and 48 h post-injury. Asterisk indicates lesion site. (B)Representative MAP-2 (red) and DAPI (blue) staining reveals the effects ofMP on neurons in the saline-treated group and the MP-treated group. Asteriskindicates lesion site. (C) The axonal dieback distance from initial lesionsite after hemisection SCI in the saline-treated SCI group (n = 6), theMP-treated SCI group (n = 6) and the sham group (n = 6). Fifteen to twentyaxons were measured per animal. Values presented are mean ± SEM.*P < 0.01. Repeated measure ANOVA followed by Fisher'sLSD. (D) The number of neurons at the edge of lesion site in saline-treatedgroup (n = 6) and MP-treated group (n = 6). Values presented are mean± SEM. *P < 0.01, P = 0.007. Statistical comparisionwas done using Student's t test.

MP increased regional microvascular blood flow and reduced microvesselloss

We used in vivo two-photon imaging of microvessels proximal to the lesionsite (Fig. 3A) to measure microvascular blood flowvelocity and vascular lumen diameter at different time point (Fig. 3B) and investigate the effect of MP treatment after SCI18,19. Our results showed that the blood flow velocity inthe sham group (n = 6) remained stable during all imaging sessions after surgery(Figs 3C and 3F). The regionalspinal cord blood flow velocity decreased progressively after hemisection SCI inthe saline-treated SCI group. The respective microvascular blood flow velocityat 30 min, 60 min, 90 min and 120 min post-injury were 1635.01 ±568.47 μm/s, 1435.77 ± 566.32 μm/s, 1175.82± 455.23 μm/s and 1074.92 ± 399.64μm/s in the saline-treated SCI group (n = 6). However, the regionalspinal cord blood flow exhibited a sustained increase post-injury in theMP-treated SCI group. The respective microvascular blood flow velocity at 30min, 60 min, 90 min and 120 min post-injury were 1734.35 ± 583.99μm/s, 2192.54 ± 593.66 μm/s, 2452.28± 535.59 μm/s and 2499.34 ± 579.88μm/s in the MP-treated SCI group (n = 6). The regional spinal cordblood flow velocity was significantly higher in the MP group than in the salinegroup (Fig. 3F, P < 0.05). However, thevascular lumen diameter in all groups exhibited no significant changes at 30min, 60 min, 90 min and 120 min post-injury (P > 0.05, Fig. 3E). We also examined the number of microvessels at theedge of lesion site at 3 days post-injury (Fig. 3D). Thenumber of microvessels were 167.2 ± 12.65 vessels/mm2in the saline-treated group (n = 6) and 231.8 ± 10.86vessels/mm2 in the MP-treated group (n = 6). The saline groupexhibited greater blood vessel loss than the MP group (Fig. 3G,P = 0.008). These results suggest that MP treatment can amelioratemicrocirculation by increasing regional microvascular blood flow and reducingmicrovessels loss, which may contribute to the attenuation of progressive axonaldamage and neuronal death.

Figure 3
figure 3

MP increased regional microvascular blood flow and reduced microvessel lossafter SCI.

(A) Representative image showing axons (green) and blood vessels (red) afterhemisection SCI. The yellow square denotes: the monitored microvessel at theedge of lesion site. (B) In vivo two-photon imaging of a microvesselproximal to the lesion site at 30 min, 60 min, 90 min and 120 minpost-injury. (C) In vivo two-photon imaging of regional microvascular bloodflow from the sham group, saline-treated SCI group and the MP-treated SCIgroup at 30 min, 60 min, 90 min and 120 min post-injury. (D) Representativeimages depict blood vessels (red) at the edge of lesion sites insaline-treated group and MP-treated group. Nuclei were stained with DAPI(blue). Asterisk indicates lesion site. (E) Changes of vascular lumendiameter in the sham group (n = 6), saline-treated group (n = 6), andMP-treated group (n = 6) at 30 min, 60 min, 90 min and 120 min post-injury.P > 0.05. (F) Changes of microvascular blood flow velocity in thesham group (n = 6), saline-treated group (n = 6) and MP-treated group (n =6) at 30 min, 60 min, 90 min and 120 min post-injury. Values presented aremean ± SEM. *P < 0.01. Repeated measure ANOVA followedby Fisher's LSD. (G) The number of microvessels at the edge oflesion site in saline-treated (n = 6) and MP-treated (n = 6) mice. Valuespresented are mean ± SEM. *P < 0.01.Student's t test.

MP reduced calcium influx and the expression of active calpain-1 andcleaved caspase-3

We used two-photon microscopy and Thy1-GCaMP transgenic mice to image the levelof intracellular calcium [Ca2+]i with agenetically-encoded calcium indicator GCaMP in injured axons in order to assessthe effect of MP on calcium influx after SCI (Fig. 4A).Changes in [Ca2+]i were expressed as changes influorescence intensity20. The level of[Ca2+]i in the sham group (n = 6) remained low andhad no significant change at 30 min, 60 min, 90 min and 120 min post-surgery (P> 0.05). Changes in the level of [Ca2+]ifluorescence in the saline group (n = 6) at 30 min, 60 min, 90 min and 120 minwere 1.89 ± 0.73, 2.51 ± 0.97, 2.87 ± 0.74,3.05 ± 0.81 respectively. However, changes in the level of[Ca2+]i fluorescence in the MP group (n = 6) at 30min, 60 min, 90 min and 120 min were 2.05 ± 0.62, 1.17 ±0.74, 0.69 ± 0.58, 0.66 ± 0.54 respectively. The[Ca2+]i in MP group were significantly lower thanthe saline group at 60 min, 90 min and 120 min post-injury (Fig.4C, P < 0.05). In order to assess the expression ofcalpain-1 gene and its apoptotic pathways that are downstream of increased[Ca2+]i, we measured changes in active calpain-1and cleaved caspase-3 post-injury by Western blots analysis (Fig.4B). Compared with saline treatment, MP treatment down-regulated theexpression of calpain-1 and active caspase-3 in injured spinal cord segments(Fig. 4D, P < 0.05).

Figure 4
figure 4

MP treatment reduced calcium influx and the expression of active calpain-1and cleaved caspase-3 after SCI.

(A) In vivo two-photon imaging of calcium influx in the sham group andinjured axons in the saline-treated SCI group and the MP-treated SCI groupat 30 min, 60 min, 90 min and 120 min post-injury. (B) Western blots ofcalpain-1 and cleaved caspase-3 expression in injured spinal cord segment ofsaline-treated group and MP-treated group. (C) Changes of calcium influxafter hemisection SCI in sham group (n = 6), saline-treated (n = 6) andMP-treated (n = 6) mice at 30 min, 60 min, 90 min and 120 min post-injury.Values presented are mean ± SEM (P < 0.05).Statistical analysis: repeated measure ANOVA. Fifteen to twenty axons weremeasured individually per animal. (D) Quantification of protein band densityto determine levels of active calpain-1 and cleaved caspase-3. *P< 0.01. Student's t test.

MP inhibited the accumulation of microglia/macrophages and down-regulatedthe expression of iNOS, MCP-1 and IL-1β

We examined the number of microglia/macrophages and MP's effect ontheir accumulation at the lesion sites 3 days post-injury (Fig.5A). The number of microglia/macrophages at the lesion site was 83.69± 9.06 cells/mm2 in the saline-treated SCI group (n =6) and 46.67 ± 6.41 cells/mm2 in the MP-treated SCIgroup (n = 6). The number of microglial/macrophages was greater in the salinegroup than in the MP group (Fig. 5B, P = 0.007). Toevaluate the anti-inflammatory effect of MP in injured spinal cord, we performeda quantitative analysis of well-known proinflammatory markers iNOS, MCP-1,IL-1β in injured mouse spinal cord removed 72 h post-injury. Strongreductions of all tested markers were observed in the MP group (n = 5) comparedwith the saline group (n = 5). iNOS expression was reduced 10.3 fold, MCP-1expression was reduced 3.6 fold and IL-1 expression was reduced 4.9 fold.(Fig. 5C, P < 0.01 for all).

Figure 5
figure 5

MP inhibited the accumulation of microglia/macrophages and down-regulatedthe expression of iNOS, MCP-1 and IL-1β after SCI.

(A) Representative F4/80 (green) and DAPI (blue) staining depicts the effectsof MP on microglia/macrophages at the lesion site in saline-treated SCIgroup and MP-treated SCI group. (B) The number of microglia/macrophages atthe lesion sites of saline-treated (n = 6) and MP-treated (n = 6) mice.Values presented are mean ± SEM. *P < 0.01.Student's t test. (C) Relative changes (quantitativereal-time PCR) of selected proinflammatory markers including nitric oxidesynthase (iNOS), monocyte chemoattractant protein 1 (MCP-1) and interleukin1 beta (IL-1β) in the injured spinal cord segments fromsaline-treated (n = 5) and MP-treated (n = 5) mice. *P < 0.01.Student's t test.

MP improved the recovery of behavioral function

To evaluate the effects of MP in behavioral function after SCI, Basso Mouse Scale(BMS) was used to assess functional improvement of all groups at different timepoints (0 D, 3 D, 7 D, 30 D, 60 D, 90 D) after surgery (Fig.6). The mice in the sham group (n = 6) exhibited mild trunkinstability (BMS score 8) on day 3 post-surgery and recovered to normal trunkstability from day 7 onward (BMS score 9). The mice in the saline-treated groupand MP-treated group exhibited no ankle movement and complete hind limbparalysis after hemisection SCI (BMS score 0). A few mice were capable of slightankle movement during D7 post-injury in saline and MP groups, but there was nosignificant difference in BMS score (P > 0.05). The respective BMSscores at 30D, 60D and 90D post-injury were 1.17 ± 0.98, 1.51± 1.22, 1.83 ± 1.16 in the saline-treated SCI group (n =6) and 2.51 ± 1.05, 3.16 ± 1.16, 3.50 ± 1.04in the MP-treated SCI group (n = 6). The BMS score was significantly higher inMP-treated group than in saline-treated group from 30 D, 60 D and 90 D afterSCI (P < 0.05).

Figure 6
figure 6

MP improved the recovery of behavioral function after SCI.

Basso Mouse Scale (BMS) scores of mice in the sham group (n = 6),saline-treated group (n = 6) and MP-treated group (n = 6) at different timepoints (0 D, 3 D, 7 D, 30 D, 60 D, 90 D) post-surgery. The BMS scores inMP-treated group were higher than saline-treated group from 30 D to 90 Dpost-injury. Values presented are mean ± SEM. *P <0.05. Repeated measure ANOVA followed by Fisher's LSD.

Discussion

Spinal cord injury includes primary and secondary injury phases. The primary injuryphase comprises immediate cell death and vascular dysfunction and is followed by adelayed secondary injury phase that can last from hours to weeks. Secondary injurytriggers a wide range of down-stream pathological events that aggravate the primaryinjury and causes progressive cell damage that is not involved in the primaryinjury21. However, the early pathological changes of axonaldieback, blood flow and calcium influx into axons in vivo after SCI remainunclear. To explore the pathogenic mechanism of SCI, we conducted this study toinvestigate the early pathological changes of axonal dieback, blood flow and calciuminflux into axons in vivo after SCI.

As the standard effective therapeutic agent now in use for the clinical treatment ofacute SCI, the glucocorticoid drug MP has been shown to alleviate secondary injuryby decreasing inflammation and ischemic reaction, as well as by inhibiting lipidperoxidation22. However, high-dose MP can cause many sideeffects, including infection, pneumonia, bleeding and femoral head necrosis, andthus increase the risk of death23,24. In addition, someretrospective cohort studies have shown no differences in neurological outcomebetween SCI patients with or without MP therapy14. The use ofhigh-dose MP in SCI patients is controversial on the basis of the risk of seriousadverse effects and modest neurological benefit. In clinical treatment for SCIpatients, MP is recommended to be administered within 8 h post-injury25. Previous study indicated that MP therapy on SCI model had a very shorttherapeutic window, the delayed treatment of MP showed no effect compared to thesaline-treated group26. In the present study, MP was initiallyadministered at 30 min post-injury and continuous administered at 6 h and 24 h toprovide an effective concentration during the first day after SCI. Our studyconfirmed that the early application of MP was effective at reducing the post-SCIdamage during the early stage and improved functional recovery at the later stage.These results consisted with previous study that MP treatment improved axonalsurvival and sprouting in complete transection SCI model27.

Previous laboratory studies of SCI were mostly confined to vitro experimentaltechniques, including tissue sectioning, immunohistochemistry and BDA labeling28. These methods do not allow us to determine dynamic changes inthe same animal over multiple days after SCI. The in vivo imaging techniquesused in the past include MRI, micro-CT, diffusion tensor tractography29, any of which can be used to examine the same animal for a couple of days.However, these methods lack resolution at the micrometer level. Recent, two-photonmicroscopy has been used to examine pinprick-induced or laser-induced SCImodels30,31,32,33,34. These models are able to controlthe damage in axons without damaging the neighboring neurons and vessels. In thepresent study, we used a hemisection SCI model, in which axons, neurons and vesselscan be damaged. We also modified a spinal stabilization device and implanted windowthat reduces the movement artifacts caused by heartbeat and breathing (Fig. 1), allowing us to examine axonal dieback, regionalmicrovascular blood flow and calcium influx into axons in the same animal formultiple hours. This in vivo imaging method allows us to evaluate the earlydynamic changes and MP's effect after SCI in a less invasive manner.

Although previous study showed that MP therapy may reduce lesion volume afterSCI35, the mechanisms underlying MP therapy remain unclear.In the present study, we conducted two-photon microscopy and employed YFP H-linetransgenic mice to trace axonal dieback after hemisection SCI. Our results indicatedthat the axons in the sham group remained intact during all imaging sessionspost-surgery. This finding indicated that the window implantation on the spinal corddid not cause significant damage to the axons. In the hemisection SCI groups, MPtreatment reduced axonal dieback distance at 48 h post-injury when compared with thesaline-treated mice. The histology revealed that the MP group also had a higherneuronal number than the saline group. In addition, MP improved the functionalrecovery at the later stage of SCI. These findings suggest that MP therapy may helpattenuate progressive axonal damage and neuronal death, improve neurologicalrecovery after SCI. These findings supported the idea that the early application ofMP improved the neuronal viability and promoted neurite outgrowth after SCI36,37.

Previous studies often used Doppler ultrasound to evaluate the blood flow afterSCI4. It is difficult to detect the microvascular bloodflow at the edge of injured epicenter using this method. In this study, we conductedin vivo two-photon imaging of microvessels of 10–20μm diameter labeled with Texas Red dextran and measured the blood flowvelocity for several hours post-injury. Our results revealed that the microvascularblood flow velocity and vascular lumen diameter in the uninjured sham group remainedstable during all imaging sessions after surgery, this finding suggested that theimplanted window on the spinal cord did not cause significant damage to themicrovessels and the blood flow. In addition, the microvascular blood flow velocityin saline-treated group decreased progressively post-injury. Thrombus, anddysfunction of vascular homeostasis might be important contributors to thisevent38. However, microvascular blood flow velocity wassignificantly increased in the MP-treated group compared with the saline-treatedgroup. These results consisted with the previous findings that MP treatment afterSCI improved microvascular perfusion39. However, the vascularlumen diameter in all groups exhibited no significant changes at all imagingsessions post-injury. Thus, the increase blood flow is not due to vasospasm andvasodilatation-induced hyperemia in the monitored venules. Histology also showedthat MP-treated mice had a higher microvessels number at the edge of lesion sitethan saline-treated mice, which suggests that high-dose MP treatment reducesmicrovessels loss after SCI.

The initial trauma in the spinal cord disrupts the cell membrane and axolemma,leading to a sudden influx of extracellular calcium. It also causes mitochondrialdamage that can affect Na-K-ATPase activity, as well as an increase of intracellularcalcium via dysfunction of Na-Ca-exchanger40. The intracellularcalcium concentration activates the calcium-activated neutral proteinase calpain-1,which results in neuronal disintegration and apoptosis41. Theseare essential pathogenic factors in the secondary phase of SCI. To understand how MPaffects calcium influx in injured axons, we used Thy1-GCaMP transgenic mice, whichexpress genetically encoded calcium indicators in neurons and axons. Two-photonmicroscopy was used to image the calcium influx in injured axons post-injury. MPtreatment produced a significant reduction of calcium influx compared with thesaline-treated group post-injury. The expression of active calpain-1 and cleavedcaspase-3 were down-regulated in MP-treated mice compared with saline-treated mice.These findings may suggest that the membrane-stabilization effects of MP preventexcessive calcium influx into cells42. MP also reduced theexpression of active calpain-1 and cleaved caspase-3 post-injury43. These changes of expression might be the important factors of how MPreduces secondary injury after SCI.

Microglia are the resident immune cells in the spinal cord. When traumatic damage isinflicted on the spinal cord, the blood-spinal cord barrier is damaged.Microglia/macrophages were recruited and accumulated at the lesion site after SCIand secreted proinflammatory cytokines that cause neuronal toxicity44,45,46. The proinflammatory mediators iNOS, MCP-1, andIL-1β are strongly associated with neurologic impairment. NO and ATPmediated the conversion of microglial shape from ramified to ameboid indicatingcellular activation33. Activated microglia/macrophages inducedaxonal dieback through direct physical interactions47. In thisstudy, we found that microglia/macrophages accumulated at the site of injury afterSCI and MP treatment inhibited the accumulation of microglia/macrophages,down-regulated the expression of iNOS, MCP-1 and IL-1β. This also mightbe a major point of the mechanism underlying the beneficial neuroprotective effectof MP in this model of acute SCI.

In conclusion, our data demonstrate that MP exerts a protective effect during theearly stages of hemisection SCI in this mouse model. Our findings are consistentwith previous studies that MP therapy may alleviate the progressive damage of axonsand reduce accumulation of microglia/macrophages48. However, weused hemisection injury model rather than compression injury model. The differenceof the injury model might be a major reason which caused different results. Inaddition, we observed a longer period to assess functional improvement of theanimals and found that MP treatment improved the recovery of behavioural functionafter SCI. Our results further suggest that MP increase microvascular blood flow andreduce microvessel loss, reduce calcium influx and down-regulate the expression ofactive calpain-1 and cleaved caspase-3 and down-regulate the expression of iNOS,MCP-1 and IL-1β. These findings suggest that early application of MP maybe an effective treatment for acute SCI.

Lastly, it is important to point out some limitations of our studies related torepeated imaging with in vivo two-photon microscopy. There was mildinflammatory responses caused by the implanted window as previous described byFarrar and our preliminary experiment49. Previous studiesshowed that even a minimal injury to the spinal cord caused enormous increase inmicroglia number and density around the lesion site. However, this increase farexceeded the microglia response caused by implanted window. This moderateinflammatory reaction does not seem to significantly affect the results caused by MPtreatment after SCI. In present study, all animals were treated in the samecondition and experienced the same model, this could help to minimize the variancebetween groups. In addition, the two-photon microscopy can only image axons lessthan 200 μm deep in the dorsal columns, it is difficult to image thedeeper tissue in live mouse spinal cord. The growth of granulation tissue alsoaffect the quality of image34. Furthermore, there are a numberof effects of MP on spinal cord injury treatment. It is not clear which of these isresponsible for the therapeutic effect. Further research needs to address theseissues.

Methods

Animals

Animal surgical procedures were conducted with the approval of the AnimalExperimentation Ethics of the Chinese PLA General Hospital. All experiments werecarried out in accordance with Animal Experimentation Ethics Guidelines of theChinese PLA General Hospital. Animals had free access to food and water. Twolines of transgenic mice, the YFP H-line and the Thy1-GCaMP line (male,8–10 weeks of age, 20–25 g) were used in this study. YFP-Hline mice specifically expressed yellow fluorescent protein (YFP) in motor andsensory neurons and axons50. Thy1-GCaMP transgenic miceexpressed a genetically encoded GFP-based calcium indicator protein in motor andsensory neurons and axons17,51. We implanted the glasswindow after laminectomy or hemisection injury to the spinal cord. Then werandomly divided each mouse line into three groups (n = 6 mice per subgroup).The sham group, the saline-treated SCI group and the MP-treated SCI group eachincluded YFP-H line mice (n = 6 per group) and Thy1-GCaMP mice (n = 6 pergroup). The sham group received laminectomy only. The saline-treated SCI groupreceived saline intraperitoneally at 30 min, 6 h, 24 h after SCI. The MP-treatedSCI group received MP intraperitoneally at 30 min, 6 h, 24 h after SCI. MP wasadministered at doses of 30 mg/kg, as recommended by the National Acute SpinalCord Injury Study (NASCIS) 2, 3 trials and as reported previously13,25,52. The criteria for animal exclusion. During thesurgery process, two YFP-H line mice died due to inappropriate anesthesia, weadded other two YFP-H line mice (male, 8–10 weeks of age,20–25 g) and randomly divided into the groups.

SCI model and implantation of the imaging window

We performed all surgical procedures with special attention to sterileconditions. 20 mg/ml ketamine and 2 mg/ml xylazine were administeredintraperitoneally to anesthetize the mice. For each moue, the dorsal surfaceabove the thoracic spinal region was shaved with an electric razor and washedwith 70% (v/v) ethanol and iodine to reduce the risk of infection. We made alongitudinal incision in the skin at the T11-T13 level of the spine and removedthe muscle and tendon tissue from the spinal arcs. After the laminectomy at thelevel of the T12 segment, we used a sharp scalpel to make a hemisection injuryin the spinal cord as previous reported53,54. Briefly, weused stainless clamps of stereotaxic apparatus to immobilize the spinal column,then we used microsurgical forceps and microscissor to tear the dura of thespinal cord segment. A sharp scalpel of 150 μm width was used to cutto the ventral cord on the middle of the spinal cord and then transected thewhole left spinal cord to the lateral side55. The averagewidth of the induced injury was 160.8 ± 7.3 μm. All thesurgery procedure were performed under the stereomicroscope. The surgicalmanipulation is very reproducible and all the SCI surgeries in present studywere performed by the same experienced operator, this could also help tominimize the variation of the lesion size. Because the bleeding was a seriousconcern for two-photon imaging process, we avoided to damage the dorsal centralvein in this model. However, there was bleeding from the injured microvesselsafter hemisection spinal cord injury. In order to avoid the influence of bloodin two-photon imaging process, we cleared the blood from the injured spinal cordby flushing the exposed cord with sterile PBS. After clearing the blood from thelesion site, we implanted a glass window on the mouse spinal cord according topreviously described methods49. Briefly, we used two metalbars to clamp the three vertebrae on either side of the laminectomy, put the topplate onto the metal bars and sealed the bone and bars with cyanoacrylate anddental acrylic. Then we applied a layer of silicone elastomer over the spinalcord and placed a glass coverslip over the spinal cord. Finally, we glued thewindow with dental cement and sutured the skin to the top plate (Fig. 1). The process of window implantation took 23.4 ±3.5 min after the hemisection injury by an experience operator. After theoperation of injury model and window implantation, we randomly divided theanimals into different groups without knowing the exact size of the injury andthen took the animals to the two-photon microscopy for the first imagingsession, we set the 30 min post-injury as the first imaging time in all groups.Postoperatively, mice were kept in a warning pad for several hours until theyregained consciousness. We manually voided the bladders of the mice twice dailyuntil voluntary control returned56. An antibiotic(enrofloxacin, 2.27 mg/kg, Baytril, Bayer, KS, USA) was used once daily for 3days. The mice had free access to food and tap water and were maintained on a 12h light/dark cycle at 22°C ± 1°C.

In vivo imaging of axonal dieback, regional microvascular blood flow, andcalcium influx into axons after SCI

To reduce motion artifacts, we positioned each mouse in a customized spinalstabilization device and slightly elevated it to allow room for breathing andchest expansion, as previously described57. We used anOlympus FluoView FV1000 two-photon microscope with an Olympus 25 ×1.0 NA water-immersion objective lens. A Spectra Physics Mai-Tai IR laser wastuned to 920 nm for two-photon excitation of YFP and to 890 nm for calciumimaging. Each mouse was kept warm at 37°C during the imaging period.The axonal dieback was a relatively slower event, so we selected the time pointat 30 min, 8 h, 24 h and 48 h post-injury to perform two-photon imagingstudies, with the blood vessels labeled with Texas Red dextran (70 kDa) asprevious described58. Fifteen to twenty axons were measuredper animal. We imaged the regional microvascular blood flow as previousreported59. After injection of Texas Red dextran intothe tail vein, we first mapped the vasculature at the edge of lesion site with a25 × 1.0 NA water-immersion objective lens. Changes in blood flow andcalcium influx was most drastic in the first 2 h post-injury, so we selected thetime point at 30, 60, 90 and 120 min post-injury to detect these events withthe hope to detect the effect of our pharmacological manipulation. We monitoredmicrovessels of 10–20 μm diameter within 200 μmof the lesion site. Linear scanning along the length of the center of eachmicrovessel was used to measure the velocity of Red Blood Cells (RBCs) at 30min, 60 min, 90 min and 120 min post-injury. The RBCs velocity was calculatedfrom the angle of the RBCs streaks60. The vascular lumendiameter was measured by the width of the vessels at 30 min, 60 min, 90 min, and120 min post-injury18. For calcium imaging, we usedThy1-GCaMP transgenic mice to investigate intracellular calcium levels ininjured axons at 30 min, 60 min, 90 min and 120 min after SCI. The laser powerat the back aperture of the objective was 30 mW at 900 nm at specimen and thepower was constant during all imaging sessions. We measured the fluorescenceintensity changes in intracellular calcium levels to evaluate the calcium influxpost-injury. Values presented are mean ± SEM. Repeated measure ANOVAfollowed by Fisher's LSD.

Image processing and quantification

Image analysis was performed using NIH Image J software. We pseudo-colored andenhanced the contrast of images to increase clarity. We traced the dieback ofindividual axons in the caudal area. We tracked axons from boththree-dimensional stacks to determine the distance between individual axon tipsfrom the initial lesion site. Fifteen to twenty axons were measured per animalto determine the average axonal dieback distance from the edge of the observedinjury49. The measurements from all animals in eachgroup were averaged to yield the average dieback distance per time point. Toevaluate changes of the regional microvascular blood flow velocity, we measuredthe velocity of RBCs with linear scanning along the length of center of eachmicrovessel and then calculated the angle of the RBCs streaks. To evaluatecalcium influx in the injured axons, we imaged the calcium fluorescenceintensity at injured axons in Thy1-GCaMP mice. Changes in[Ca2+]i were expressed as changes in fluorescenceintensity. (F―F0)/ F0 was used where we defined F as the fluorescencein single axon and F0 as the resting fluorescence signal20.Fifteen to twenty axons 200 μm away from the lesion edge weremeasured individually per animal.

Histology

We performed a histological analysis at the lesion site 3 days post-injury.Animals were deeply anesthetized and perfused transcardially with 20 ml PBSsolution, followed by fixation with 20 ml 4% paraformaldehyde (PFA). We immersedthe entire spine in 4% PFA for 1 day and then removed the spinal cord from thevertebral canal with microsurgical scissors. The spinal cord was immersed in 30%sucrose until saturated and embedded into optimal cutting temperature (OCT)compound. We froze the spinal cord at g−80°C overnight andcut 10 μm sections on a Microm HM 525 cryotome (ThermoFisherScientific). We blocked with a mixture of 2% goat serum in PBS for 1 h. Next,sections were incubated overnight at 4°C with a primary anti-MAP2 IgGantibody (1:100 dilution; Millipore, USA), anti-F4/80 IgG antibody (1:100dilution; Biolengend, USA). After incubation with the primary antibody, werinsed tissue sections in PBS and incubated them with FITC-conjugated anti-mousefluorescent secondary antibodies (1:100 dilution) for 2 h and then incubatedwith DAPI at room temperature for 20 min. Blood vessels were directly labeled bydye DiI (Sigma, USA) as previous reported61. Briefly, 100mg of DiI crystal was dissolved in 16.7 ml of 100% ethanol. After deeplyanesthetized the mice, we perfused the mice transcardially with 5 ml DiIsolution at a rate of 1–2 ml/min. In this method, DiI molecules weredirectly incorporated into the membrane of endothelial cells. After DiI stainingthe blood vessels, the spinal cord was fixed and cut sections as abovedescribed. We examined the sections with a fluorescence microscope. Neuronalsomata were manually counted based on the morphology of neuron andcounter-staining of MAP-2 antibody (red) as well as DAPI (blue). Microvesselswere manually counted based on the counter-staining of DiI (red) as well as DAPI(blue). We counted the neurons and microvessels in two rectangular area (0.39mm2) at the edge of lesion site per section. Three sectionsper mouse were quantified. Microglia/macrophages were manually counted based onthe morphopogy of microglia/macrophages and counter-staining of F4/80 antibody(green) and DAPI (blue). Microglia/macrophages were counted in a rectangulararea (5.07 mm2) in the middle of lesion site per section. Threesections per mouse were quantified. Six mice in each group were used.

Protein extraction and Western bolt analysis

Western blots was performed as reported previously62. Weharvested and froze the injured spinal cord tissue at−70°C and then homogenized the tissue in buffer containing50 μM Tris-HCl (pH = 7.4), 1 mM phenylmethysulfonyl (PMSF; BethesdaResearch Laboratories, Gaithersburg, MD, USA) and 5 mM EGTA (Sigma) andhomogenized with a Polytron batch homogenizer. We centrifuged the homogenizedsamples in an Optima LE-80K Ultracentrifuge (Beckman Coulter, Fullerton, CA,USA) for 1 h at 100,000 g. After centrifugation, we mixed protein samples withsample buffer and then boiled for 5 min and stored at−20°C. We loaded the samples onto 20% gels andelectrophoresed at 200 V for 30 min. We then resolved the proteins and used aGenie transfer apparatus to transfer the samples to nylon membrane. We blockedthe nylon membrane for 1 h in 5% nonfat milk in Tris/Tween buffer. We incubatedthe membranes overnight with primary IgG antibody (1:5000anti-β-actin (clone AC-15; Sigma), 1:500 anti-active calpain-1(Abcam, USA) and 1:500 anti-cleaved caspase-3 (Cell Signaling, USA)). Weincubated the membranes with donkey anti-rabbit secondary antibody (diluted1:2000; Biolegend, USA) for 1 h after washing three times with Tris/Tweenbuffer. We then incubated the membranes with chemiluminescent (ECL) reagent(Amersham, Piscataway, NJ, USA) and exposed them to X-Omat AR films (Kodak,Rochester, NY, USA). We scanned the films on a Umax PowerLook Scanner and usedPhotoshop software (Adobe Systems, Seattle, WA, USA) for image processing. Weused Quantity One software (Bio-Rad) to determine the optical density (OD) ofeach band40,62.

RNA extraction and real-time PCR analysis

We used the RNeasy Mini Kit (Qiagen, Germantown, MD, USA) to isolate the totalmRNA from injured spinal cord segments (1 cm containing and surrounding thelesioned area) 3 days after SCI. One milliliter Trizol (Life Technologies) wasused to homogenize the tissues and RNA was extracted according to themanufacturer's protocol. We synthesized cDNA from 1 μgtotal RNA using iScript cDNA synthesis Kit (Bio-Rad, Hercules, CA, USA) aftertreatment with DNase (Promega, Madison, WI, USA). We used SYBR-Green basedtechnology to perform real-time PCR in the CFX Connect Real-Time PCR DetectionSystem (Bio-Rad); the following primers were used: nitric oxide synthase 2(iNOS2) (Fw: AAACCCCAGGTGCTATTCCC; Rv: GAACATTCTGTGCAGTCCCA); monocytechemoattractant protein 1 (MCP-1):( Fw: ACGCTTCTGGGCCTGTTGTT; Rv:CCTGCTGCTGGTGATTCTCT); Interleukin-1 beta (IL-1β): (Fw: TGGCAACTGTCCCTGAACTC; Rv: GTCGAGATGCTGCTGTGAGA). We analyzed the data using Bio-Rad CFXManager 3.0 (Bio-Rad). The gene glyceraldehyde-3-phosphate dehydrogenase (GADPH)was chosen as reference. The mRNA level of each target gene was normalized byGADPH and expressed as 2ΔCt (ΔCt = Cttarget -Ct GADPH). The relative quantity in mRNA levels of tested genes wasdetermined by the equation: relative quantity =1000/2ΔCt.

Behavioral testing

We used the Basso Mouse Scale (BMS) score to assess functional recovery after SCIas previously described63. Hind limb motor function wasassessed with the 10-point scale in an open field. No ankle movement andcomplete hind limb paralysis scored 0; Slight ankle movement scored 1; Mildtrunk instability scored 8 and no locomotor deficits scored 9. We assessed andscored the functional improvement of all groups on 0 D, 3 D, 7 D, 30 D, 60 D, 90D after surgery. All experiments were performed in a double-blind manner. Valuespresented are mean ± SEM. Repeated measure ANOVA followed byFisher's LSD.

Statistical analysis

The statistical analysis was performed using SPSS (version17, SPSS IL, Chicago).Data are presented as mean ± SEM. We compared axonal diebackdistance, microvascular blood flow, vascular lumen diameter and calcium influxinto axons using repeated measure ANOVA followed by Fisher's LSD. Wecompared number of neurons, microvessels, protein expression,microglia/macrophages and inflammatory factors using Student'st test. Significant differences were defined at P < 0.05.Highly significant differences were defined at P < 0.01.

Ethical statement

Animal surgical procedures were conducted with the approval of the AnimalExperimentation Ethics of the Chinese PLA General Hospital. Care was taken tominimize the number of animals used and their suffering.

Additional information

How to cite this article: Tang, P. et al. In Vivo Two-Photon Imaging ofAxonal Dieback, Blood Flow and Calcium Influx with Methylprednisolone Therapy afterSpinal Cord Injury. Sci. Rep. 5, 9691; DOI:10.1038/srep09691 (2015).