| Background:Peripheral nerve injury is a common and challenging disease worldwide,with an incidence of about 3%of all traumatic injuries and about 20 million people with various degrees of combined neuropathy in the world.The prolonged and expensive treatment of peripheral nerve injuries has significantly reduced the patients’quality of life and burdened them financially and psychologically.Despite almost a century of research for peripheral nerve damage,the present outcome of neurological recovery is still unsatisfactory.Lithium salts were a widely used mood stabiliser in clinical practice for over 60 years.In recent years,a growing evidence has shown its broad neuroprotective effects and therapeutic potential for neurodegenerative diseases(e.g.Parkinson’s disease,Huntington’s disease,Alzheimer’s disease,etc.),traumatic and ischemic brain injury disorders,and motor neuron related disorders such as lateral sclerosis of the spinal cord.Besides this,lithium has also been reported to promote the recovery of peripheral nerve injury,but the mechanism is still unclear.Lithium salts have excellent biological properties,however,the prescription of lithium salts is decreasing annually.There are two main reasons for this paradox:firstly,lithium has very close effective therapeutic and toxic concentrations(extremely narrow therapeutic window),and the use of lithium salts in clinical work requires strict indications and tight testing of blood levels.This carries certain medical risks as well as increasing the patient’s burden.Secondly,lithium,the smallest metal ion with the smallest molecular weight,has a structure similar to sodium ions and could competitively bind sodium channels and lead to a range of side effects.Based on this,we initially investigated whether lithium citrate(LC)could promote the repair of damaged sciatic nerve in rats and explored the possible mechanisms in animal experiments.We subsequently synthesized Li~+-doped carbonized polymer dots(Li-CPDs)by a one-step hydrothermal method to avoid Li~+competition for sodium channels while preserving the biological function of lithium ions.Following the successful synthesis of Li-CPDs,we explored the biological compatibility and whether they were sciatoprotective at the cellular and animal levels.Purpose:(1)To construct a rat sciatic nerve injury(SNI)model and verify whether LC can promote the repair of the injured rat sciatic nerve based on the constructed model.(2)To prepare and characterize Li-CPDs,and to examine the biocompatibility and cellular uptake patterns of Li-CPDs.To verify the nephrotoxicity of LC and Li-CPDs after short-term high dose administration.(3)To validate the biological function of Li-CPDs.Investigate the effect of Li-CPDs on the recovery of injured sciatic nerve function in animal studies and explore the potential mechanism of Li-CPDs to promote nerve regeneration.Methods:(1)Establishment of SNI model and verification of the therapeutic effect of LC.Rats were randomly divided into two groups:the control group local injection of PBS after SNI(n=18);and the LC group with local injection of LC after SNI(lithium citrate group,n=18).The rat SNI model was established via the crush method.7 days after surgery,the established SNI in rats was detected according to the sciatic nerve function index(SFI)and neuromuscular electrical conduction to examine whether the degree of SNI was consistent and whether the degree of injury met the experimental requirements.At 21 days postoperatively,the effect of LC on the repair of SNI was evaluated by the results of behavioural,neuromuscular electrical conduction and sciatic nerve morphology in rats.(2)Preparation and characterisation of Li-CPDs.Li-CPDs were synthesized by a one-step hydrothermal method,and the particle size,photoluminescence spectra,UV-Vis absorption spectra,X-ray photoelectron spectra,and lithium content of the material were characterized,and their biocompatibility was examined by CCK8 and AM/PI.(3)Detection of nephrotoxicity of LC and Li-CPDs.Rats were randomly divided into five groups:intraperitoneal PBS group(Control group,n=3);intraperitoneal LC group(10mg/d LC,n=3);intraperitoneal LC group(20 mg/d LC,n=3);intraperitoneal Li-CPDs group(15 mg/d Li-CPDs,n=3);intraperitoneal Li-CPDs group(30 mg/d Li-CPDs,n=3).After 2weeks of continuous administration,the nephrotoxicity of LC and Li-CPDs was evaluated by renal histomorphology and liver and kidney function results.(4)Experimentation on the repairing effect of Li-CPDs on SNI.Rats were randomly divided into four groups:the control group with local injection of PBS after SNI(n=9);the N-CPDs group with local injection of N-CPDs after SNI(n=9);the LC group with local injection of LC after SNI(n=9);and the Li-CPDs group with local injection of Li-CPDs after SNI(n=9).The rat SNI model was constructed by the crush method.7 days after surgery,the established rat SNI was examined according to the SFI and neuromuscular electrical conduction to see whether the degree of injury was consistent and whether the degree of injury met the experimental requirements.The effect of Li-CPDs on the repair of SNI was evaluated by the results of behavioral,neuromuscular electrical conduction and sciatic nerve morphology in rats at 7,14 and 21 days postoperatively.Results:(1)LC could promote the repair of motor function in rats with SNI model.At 7 days after the injury,there was no significant difference between the two groups indicating that the degree of SNI was consistent in each group;at 21 days after the injury,there was a significant improvement in the motor function of the rats in the LC treated group,indicating that the motor function of the rats in the SNI model was significantly restored after treatment with LC.The results of transmission electron microscopy(EM)showed that the number of myelinated nerve fibers increased significantly and the thickness of myelin sheath increased obviously in the rats with SNI after treatment with LC compared with the control group.This indicates that LC can promote the repair of SNI in the model rats.(2)Li-CPDs have good stability and biocompatibility.The Li-CPDs were prepared from urea and LC by a one-step hydrothermal method and were homogeneous and spherical in shape,showing green fluorescence under UV excitation.The lithium content was determined by ICP-ES of 9.56583 mg/L(0.2 mg/m L for Li-CPDs)and the survival rate of RSC96 cells incubated with Li-CPDs was higher than 80%in cellular experiments;in animal experiments,short-term intraperitoneal administration of LC at high doses resulted in vacuolisation and even apoptosis,widening of the Bowman’s capsule and bridging of the renal tubules.Compared with the control group,the plasma NAG was significantly increased after short-term high-dose application of LC.and the kidneys of rats injected with Li-CPDs did not show any significant abnormalities,indicating that Li-CPDs have good biocompatibility.(3)Li-CPDs were able to increase the autophagy flux of Schwann cells and accelerate the recovery of motor function in the SNI rats.The behavioural,histological,neurophysiological and target organ recovery of the SNI rats treated with Li-CPDs was superior to that of the control rats.The autophagy flux in Schwann cells treated with Li-CPDs was significantly higher at 7 days after sciatic nerve injury.Conclusions:Local injection of LC after SNI significantly promoted axonal regeneration and markedly improved motor function in rats.The synthesized Li-CPDs,a novel Li~+-containing nanomaterial,could avoid Li~+-induced renal injury,while increasing the intracellular autophagy flux in Schwann cells,accelerate the regeneration of the injured sciatic nerve and dramatically modify the motor function. |
