Rapid Fluorescence Imaging Of Spinal Cord Following Epidural Administration Of A Fluorescent Nanocomposite

Background:Iatrogenic spinal cord injury(SCI)is one of the most devastating complications of spine surgery,which usually results in permanent and serious disabilities of patients.However,the existing diagnostic technologies and monitoring means,such as MRI,intraoperative electrophysiological monitoring(IOM)technologies and neuronal tracing technologies,are still not satisfied for improving direct visualization of spinal cord by naked eye and visually monitoring the integrity of spinal cord during surgery.Fluorescence-guided surgery(FGS)has now become a promising technology for improving intraoperative visualization and accuracy.However,to the best of our knowledge,this technology has not been used for spine surgery.This is probably due to the difficulty to label the central nervous system(CNS)via intravenous injection of a fluorescent probe that is unable to across the blood-brain barrier(BBB).In the preliminary work,we first synthesized a florescent material—(BMB-micelle,BMB-m).Then,we demonstrated an rapid and efficient imaging of the spinal cord in rabbit via epidural administration of BMB-m.We also have found that this BMB imaging technology is revealed to hold great potential for visually monitoring the integrity of spinal cord in real time and promptly identifying the acute SCI during spine surgery.Objective:The purpose of this study is to explore the feasibility of rapid fluorescence imaging of spinal cord following epidural administration of a fluorescent nanocomposite.Meanwhile,we aim to figure out the mechanism of rapid fluorescence imaging of spinal cord.Moreover,this study aimes to investigate the application and safety of rapid fluorescence imaging of spinal cord for visually monitoring the intraoperative spinal cord injury.Methods:(1)The synthesis and physicochemical properties of BMB-m.The 1,4-bis(p-aminostyryl)-2-methoxy-benzene(BMB),a typical nerve-highlighting fluorophore,which was synthesized and firstly encapsulated into polymeric micelles(BMB-m)to form a fluorescent nanocomposite(BMB-micelle,BMB-m).The structure and spectral characteristics of BMB-m were analyzed by transmission electron microscope(TEM)and fluorescence spectrophotometer,et al.(2)BMB imaging of spinal cord in vivo.To evaluate in vivo fluorescence imaging effect of the nanocomposite,Japanese white rabbits,weighing roughly 2.5 kg were used as animal model,which received epidural administration of BMB-m in difference dosages(10,30,50 and 100 μg/ rabbit,at 30 min post-injection)or in difference times(0.25,0.5,1,2,4,8,14,and 24 h,with the same dose 50 μg/ rabbit),(n = 3 rabbits per dose or time point).Rabbits given with a single injection of blank F127 micelles,normal saline(0.9% sodium chloride solution),sham operation,untreated or intravenously infusion were used as the controls(n = 3).The spinal cord,sciatic nerve and surrounding tissues were exposed and imaged in the animal fluorescence imaging system(FIS).All fluorescent and white light images were acquired with 20 ms exposure time and identical normalizations.(3)The methods for elucidating the mechanisms of rapid fluorescence imaging of spinal cord were showed as follows: 1)Observation of the fluorescence signal at the lumbar nerve roots;2)Measurements of the fluorescence intensities and staining thickness of the cross sections at lumbar,thoracic and cervical site using confocal laser scanning microscopy(CLSM);3)Detection of BMB in CSF by RP-HPLC-ESI-MS;4)Real-time and in-situ monitoring of the fluorescence at the T10 spinal cord after a single-dose of epidural BMB.(4)Detection of spinal cord injury by BMB imaging.At 30 min post-injection(50 μg/ rabbit),two typical models,i.e.clip compressive SCI model(Rivlin method)and weight-drop SCI model(Allen method)were established at the T10 spinal cord.The white light and fluorescence images of the T10 spinal cord were obtained before and after acute injury.The fluorescence of cross sections at the injury site and neighboring non-injury sites of spinal cord were observed by the CLSM(5)The safety evaluation of the fluorescent nanocomposite after epidural administration.The toxicological effect of the fluorescent nanocomposite BMB-m were investigated by cell viability assay towards PC-12 and L929 cells,and histopathological observation of the spinal cord,main organs and tissues by HE staining.Results:(1)The BMB-m was finally obtained as yellowish solid,which improved the solubility of BMB in saline solution at different BMB concentrations ranging from 0.05 to 0.5 mg/ mL.The formed BMB-micelles(BMB-m)are of spherical structure with the size at ca.84 nm.The absorbance and emission wavelengths of BMB-m were 385 and 525 nm,respectively.(2)In the multiple dose study,the complete imaging of spinal cord can be realized even at the dose as low as 10 μg/rabbit.The fluorescence ratios of spinal cord to adipose(SC/A)of 50 μg/rabbit dose group are significantly higher than those of 10 and 30 μg/rabbit dose groups(P < 0.05),while it shows no remarkable difference with 100 μg/rabbit dose group.In addition,the SC/A at lumbar,thoracic and cervical spinal cord segments at different doses were analyzed.In the dose group of 10 μg/rabbit,the SC/A ratio of lumbar segment is significantly higher than those of thoracic and cervical segments(P < 0.05).In the dose groups from 30 to 100 μg/rabbit,the SC/A ratios of lumbar and thoracic segments are significantly higher than that of cervical segment(P < 0.05).For the kinetic study,the staining of the whole spinal cord occurred quickly at a very short time(<15 min).The brightest fluorescent imaging appeared at 30 min post-injection.After 30 min,the fluorescence on spinal cord would decrease over time and almost disappeared at 24 h.Meanwhile,the meaningful imaging(SC/A ratio > 2)of lumbar,thoracic and cervical could last up to 14,8 and 2 hours,respectively.Compared with intravenous injection group(5.0 mg of BMB/rabbit,which is 100-fold higher than that of the epidural administration group),the SC/A ratio of spinal cord in the epidural administration group(6.7)is 19.1-fold higher than that in the intravenous administration group(0.35).Furthermore,the fluorescence signals also appeared in the sciatic nerve,muscle,liver,kidney,lung,etc.in the intravenous administration group,while none of these tissues/organs were detected with visible fluorescence in the epidural administration group.(3)BMB is transported by the flow of cerebrospinal fluid(CSF),which can result in the rapid fluorescence imaging of spinal cord.The evidences were provided as follows: 1)The fluorescence signal was found to be ended at the dorsal root ganglions;2)As observed by the CLSM,the fluorescence intensity and staining thickness at the rim of spinal cord decreased from lumbar section to cervical section;3)The BMB was detectable in the CSF obtained from the cerebellomedullary cistern;4)The observation of CSF fast-flowing from caudal to cranial site on the dorsal part of spinal cord following lumbar epidural administration.(4)For both SCI model,the fluorescence at the T10 spinal cord was homogenously distributed before the injury,while darkened fluorescence could be clearly observed on the injury sites.In addition,structural changes of the white matter at the injury sites were demonstrated by the CLSM observations.(5)The BMB in both free and micellar formulation showed excellent cell biocompatibility and low cell cytotoxicities toward both PC-12 and L929 cells.The results of histopathological test showed that no pathological changes were found in the spinal cord,main organs and tissues after epidural administration in the 100 μg/rabbit dose group.Conclusions:In this study,we demonstrated the fluorescent nanocomposite(BMB-m)can be used for rapid and effective imaging of the whole spinal cord in rabbit via epidural administration.The mechanism revealed that BMB was transported by the flow of CSF and binds to the peripheral white matter,resulting in rapid staining of the whole spinal cord.Meanwhile,this technology also holds great potential for visually monitoring spinal cord integrity in real time and promptly identifying acute SCI during spine surgery.Furthermore,the results of cell viability assays and histopathological observations revealed that the fluorescent nanocomposite has good biocompatibility.