| Background and objective:Surgical resection is the main treatment strategy for skull base tumours such as trigeminal schwannoma,vestibular schwannoma and petroclival meningioma.In the process of surgical resection of skull base tumours,the cranial nerve is often difficult to differentiate from the tumour boundary due to the close adhesion,compression deformation and even complete wrapping of the lesions,which leads to iatrogenic injury of the cranial nerve.In the treatment of vestibular schwannoma,even in the world’s top neurosurgical centre,the postoperative neurological impairment(HB ≥ 3)of patients with large vestibular schwannoma still reached 56%.Similarly,30% of the patients had different degrees of facial numbness,trigeminal neuralgia,abducens nerve dysfunction and other complications after the surgical resection of trigeminal schwannoma.The occurrence of the above-mentioned nerve interference and complications after injury puts patients under great physiological,psychological and other pressures and greatly increases the social cost and economic burden of patients.Ordinary MRI can only image the tumour or brain tissue,while T2 diffusion tensor imaging(DTI)technology can use the anisotropy of water molecules in different tissues to depict the course of nerve fibre tracts.However,when the nerve structure is compressed,deformed or even destroyed by the space-occupying lesion,the imaging effect of this imaging technique will be significantly reduced.Although intraoperative neurophysiological monitoring(IONM)is the main tool to determine the structure and course of peripheral nerves,it still faces some inherent limitations in the operation process.First,the sensitivity of IONM is prone to false negatives due to the interference of muscle relaxants.Second,due to the differences in experience and subjective understanding,different operators may have obvious differences in the choice of electric stimulation quantity and stimulation site,which makes the monitoring results vary from person to person or even appear opposite from person to person.In addition,IONM has the characteristic of hysteresis;when the operator cannot analyse and predict the position and course of the nerve structure due to lack of experience,the nerve monitoring effect of IONM will be significantly reduced.In recent years,visualization surgery has been widely used in the clinic.Indocyanine(ICG)in the intraoperative fluorescein angiography of aneurysms and sodium fluorescence(FN)in fluorescent staining of gliomas in functional areas have achieved the purpose of intraoperative real-time fluorescence imaging.However,regardless of the rapid development of vascular structure or the continuous fluorescent staining of brain tumours,the specific development of the neural structure cannot be obtained in the case of tight peripheral nerve tumour adhesion,which makes it difficult to distinguish the neural structure and course in the tumour resection process.Based on practical clinical problems,our group mainly studied the synthesis of the nerve-specific fluorescent imaging agent mPLA-PEG/OX4,the establishment of a nerve tumour adhesion model and the analysis of the nerve-specific fluorescence imaging effect of the agent in a tumour model.Materials and Methods:1.Synthesis of mPLA-PEG/OX4,its phenotype and in vitro fluorescent imaging in fibres: Extraction method was applied to desalinate with perchlorate OX4.We prepared a mixture of PBS and mPLA-PEG and coated desalinated OX4 with the mixture using modified extraction method.Subsequently,we measured the diameters of composite mPLA-PEG/OX4 and identified their size distribution and Zeta potential using a DLS potentiometer.The morphology was observed by TEM.The encapsulation efficiency,encapsulation capacity and in vitro release rate were studied by UVSP.After all of these physical and chemical properties of the nanocomposite were detected,the L929 cell line with stable proliferation was established.Then,we evaluated the cytotoxicity of mPLA-PEG/OX4 on L929 cells at different concentrations.The sciatic nerve fibres of SD rats were cultured in a medium containing fluorescent imaging agent,and the effect of nerve fluorescence imaging was observed.2.Establishment of a nerve-tumour adhesion model in nude mice and fluorescence imaging of peripheral nerves: After the neurospecific fluorescence imaging function of mPLA-PEG/OX4 and OX4 was preliminarily confirmed by nerve fibre staining in vitro,a BALB/C nude mouse model of GL261 glioma of the sciatic nerve was established.Meanwhile,the sciatic nerve of 10 other nude mice was injected with an appropriate volume of PBS.The difference in fluorescence imaging effect between the sciatic nerve on the operating side and the normal sciatic nerve on the opposite side was observed 10 days later to analyse whether pathological changes such as scar formation and the inflammatory response on the nerve surface would cause significant changes in the fluorescence signal.The possibility that pathological changes at the surgical site significantly affected the effect of nerve-specific fluorescence was excluded.Then,a preliminary comparative study was conducted on the fluorescence imaging effect of mPLA-PEG/OX4 and OX4 in this model.3.Establishment of a nerve-tumour adhesion model and peripheral nerve fluorescence imaging in SD rats: First,we successfully established a nude mouse model of sciatic glioma and found that the OX4 induction by mPLA-PEG could enhance the fluorescence imaging effect of nerve structures near the tumour.Then,we established sciatic schwannoma and trigeminal schwannoma animal models in SD rats using the RT4 cell line.In this study,the sciatic nerve was not only the most frequently selected modelling object in the study of peripheral nerve tumours.The successful establishment of sciatic nerve schwannoma and characteristics of fluorescence imaging lay a foundation for the study of a trigeminal nerve schwannoma model and fluorescence imaging of nearby cranial nerves.To more vividly simulate the common clinical cranial base tumour-nerve adhesion phenomenon and the process of intraoperative nerve structure recognition,this study finally established a trigeminal schwannoma model in SD rats and compared the fluorescence imaging effect and significance of mPLA-PEG/OX4 and OX4 on cranial nerves.Before the in vivo experiment of the tumour-nerve adhesion model,we prepared sciatic schwannoma and trigeminal schwannoma tissue sections and performed a fluorescence staining experiment in vitro.To confirm the distribution characteristics of fluorescence imaging agents in tumour nerve tissues,we retained the tissues of sciatic schwannoma and trigeminal schwannoma after fluorescence imaging in vivo.Then,frozen sections were made,and the imaging characteristics of the sections were observed under a fluorescence microscope.Results:1.The synthesis,characteristics,loading capacity and release rate of mPLAPEG/OX4: mPLA-PEG/OX4 were successfully obtained by modified extraction methods.A combination of DLS and TEM provides insight into the suitable surface charge and uniform size of particles.UVSP techniques indicate that the drug-loading rate accounted for 93.3% in mPLA-PEG/OX4-and OX4-saturated aqueous solutions(10 mg/m L),and 10 mg mPLA-PEG/OX4 contained approximately 3 mg OX4.The release rate reached 70% after 72 hours with dialysis.2.Cytotoxicity of mPLA-PEG/OX4 and OX4: The cytotoxicity of 0.1 mg/m L mPLA-PEG/OX4 was significantly lower than that of 0.03 mg/m L free OX4(P <0.05),which indicates that the encapsulation process by mPLA-PEG could significantly reduce the cytotoxicity of free OX4.3.Fluorescence imaging efficacy for nerve fibres and nerve-tumour tissue sections in vitro: mPLA-PEG/OX4(experimental group)and OX4(control group)cultured in the medium had obvious neurofluorescence imaging efficacy.The sciatic nerve schwannoma and trigeminal nerve schwannoma tissue sections with staining fluorescence showed obvious nerve fluorescence imaging,while the tumour showed no fluorescence signal.There was no significant difference in fluorescent signals between the two groups.In addition,in the in vitro staining of nerve fibres and nervetumour tissue sections,fluorescent signals were not observed in the nerve and tumour tissues of the mPLA-PEG and PBS groups.4.Tumour formation rate of the nerve tumour model.In the sciatic glioma model from 38 nude mice,the tumour formation rate of 31 mice was 100% after 10 days of cell implantation,and 7 other mice died after anaesthesia.The average maximum tumour diameter was 7 mm in the sciatic glioma model.Among the 24 SD rats in the sciatic schwannoma model,only 1 of 18 rats showed no tumour formation 15 days after the cell implantation,and 6 rats died after anaesthesia.The overall tumour formation rate was 94%(17/18).The average maximum diameter of the tumour was12 mm.In the trigeminal schwannoma model in 24 SD rats,the tumour formation rate of 20 rats was 90%(18/20)after 21 days of cell implantation,excluding 4 rats that died after anaesthesia.The average maximum tumour diameter was 6 mm.5.The optimal time for fluorescence imaging of mPLA-PEG/OX4 and OX4 in vivo: In the mouse sciatic glioma model,we found that the optimal time of nerve fluorescence imaging was 3.5 h after IV both experimental group(mPLA-PEG/OX4,n=6)and the control group(OX4,n=6).The fluorescence ratio of nerves/background(NBR)was significantly higher than that at 1 h and 5.5 h after IV(P< 0.05).In addition,the NBR in the experimental group were significantly higher than those in the control group at 3.5 h after IV(P<0.05).6.Influence of the surgical operation for nerve fluorescence imaging: After the sciatic nerve of mice was injected with PBS,there is no significant difference in normal BNR in the operating and contralateral sides between the mPLA-PEG/OX4group(n=5)and the OX4 group(n=6)(P<0.05);The NBR value of sciatic nerve in the mPLA-PEG/OX4 group was significantly higher than that in the OX4 group(P<0.05).7.Fluorescence imaging of mPLA-PEG/OX4 and OX4 in vivo in a model of nerve tumour adhesion.In the nerve fluorescence imaging of the adhesion model about the nerve-tumours include sciatic neuroglioma of mice,sciatic schwannoma,and trigeminal neurinoma.The experimental group(mPLA-PEG/OX4)and control group(OX4)had significantly higher tumour-side NBR than the blank control group,and the experimental group had a significantly higher tumour-side NBR than the control group.In the fluorescence imaging of contralateral normal nerves,the experimental group had a significantly lower NBR than the control group(P<0.05).8.Distribution characteristics of fluorescence imaging agents in vivo after experimentation in nerve-tumour tissues: Similar to the above in vitro staining characteristics,fluorescence signals were mainly distributed in nerve tissue regions in the frozen nerve-tumour sections of both experimental group(mPLA-PEG/OX4)and control group(OX4).The neural fluorescence signal in the experimental group was more significantly enhanced than that in the control group,and the residual fluorescence signal in the tumour was observed in the experimental group.No fluorescence signal was observed in the tumour and nerve tissue of the blank control group.Conclusion:1.OX4 is a small fluorescent molecule that can specifically bind with peripheral nerve.It forms a nano complex with uniform particle size and moderate surface charge under the encapsulation of mPLA-PEG.2.The encapsulation effect of mPLA-PEG can reduce the cytotoxicity of OX4 and prolong the biological half-life,so as to improve its biological safety and significantly improve its nerve specific fluorescence imaging effect.3.In this study,the concept of animal model of neurotumor adhesion was used for the first time to study the real-time development of peripheral nerve.The establishment of stereotactic animal model of trigeminal schwannoma has not been reported.The survival rate and tumorigenesis rate were more than 90% in the adhesion model,which provided a model guarantee for the follow-up study of peripheral nerve tumor.4.In vivo study of nerve specific fluorescence imaging,this experiment confirmed that the mPLA-PEG / OX4 nanocomposite has more efficient and lasting fluorescence imaging function than free OX4;This phenomenon provides a theoretical basis for the recognition and protection of nerve tissue in the treatment of peripheral nerve tumors such as spinal nerve and cranial nerve. |
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