Role Of Microfluidic Chips In FK506-and Nerve Growth Factor-promote Nerve Regeneration

Acute spinal cord injury (ASCI) is a serious hazard to human health of a disease,with the continuous development of the society, and its incidence is rising, a hugeburden to the patient’s family and social. Spinal cord injury in pathological processes,including the primary spinal cord injury and secondary spinal cord injury, wherein theprimary spinal cord injury by trauma mechanism decision is irreversible damage;several hours to several days after the primary injury occurs, the injury of the spinalcord change, these pathological response and tissue of the spinal cord near a series ofpathological reactions, such as bleeding, edema, microcirculation, increased local tissueoxygen radicals such as aggravated damage to the spinal cord tissue, known assecondary injury, it generates the damage is far more than the primary injury. So earlyapplication of effective drug treatment for spinal cord injury can not be ignored.Currently, the research of drugs mainly in the following categories: corticosteroids,excitatory amino acids (EAA) receptor antagonists, neurotrophic factor (NF),antioxidants and free radical scavengers, calcium channel antagonists and other channelantagonists, opioid receptor antagonist, prostaglandin, cyclooxygenase inhibitors andspecific COX-2inhibitors. Used clinically recognized the positive effect of the drugmethylprednisolone (MP), the application of large doses of methylprednisolone withineight hours after spinal cord injury can reduce the secondary edema after spinal cordinjury, improve microcirculation, inhibition of lipid peroxidation, reducing thegeneration of oxygen free radicals, reduce calcium influx, maintain the excitability ofneurons, and promote the recovery of neurological function, so as to avoid furtherdamage of the spinal cord tissue. Methylprednisolone treatment time is limited to lessthan8hours after injury, such as spinal cord injury eight hours after application, is notonly ineffective, and there will be complications. Most of the other drugs, a link is valid only for spinal cord injury, secondary injury, spinal cord injury is the result of jointaction of a variety of mechanisms, so the treatment effect is limited. Therefore, the jointdifferent mechanism of action of drugs to treat spinal cord injuries will be the directionof future research.In recent years, with the advent of high-throughput drug screening technology, totrace drug screening, rapid and large-scale goals opened up new avenues. But it hascertain limitations, such as a porous plate (a large amount of the reagent is usuallyconsumed in a96-well plate), the sample distribution and the operation cumbersome,expensive equipment, etc.. Microfluidic chip technology based on micro-processingtechnology analysis faster, less reagent consumption, ease of integration andhigh-throughput analysis of many advantages, can overcome the limitations ofhigh-throughput drug screening, drug screening is based on the cellular level, tissueengineering research provides a new research platform.Tacrolimus (FK506) is a macrolide antibiotic with strong immunosuppressiveeffects. Recent studies have shown that protect the spinal cord injury in rats after partialnerve tissue of FK506to promote nerve regeneration, reduce neuronal apoptosis andpromote the recovery of spinal cord function. Nerve growth factor (NGF), aneurotrophic factor family (NTFS), is widely distributed in the surrounding tissue,peripheral nervous system and central nervous system. Motor neurons after spinal cordinjury can be induced NGFR expression of exogenous NGF injected into the spinal cordinjury site, to make NGF and NGFR combination, can protect neurons and promoteaxonal regeneration. The study confirmed that FK506and NGF function to promoteperipheral nerve regeneration and nutrition nerve injury have a synergistic effect, but inthe treatment of spinal cord injury if also has the same effect has not been reported.In this study, the microfluidic chip technology into drug screening system topromote nerve regeneration research areas, constructed PC12cells as effector cellspromote nerve regeneration. The best drugs combined concentration in vitro screeningFK506, NGF, and the two joint application to the treatment of acute spinal cord injuryin rats, to explore whether a synergistic effect on both functional recovery after spinalcord injury and its possible mechanism to explore. Part1Microfluidic chip design, production and PC12cell culture in the chipObjective: Build into a set of cell culture, drug intervention and protein detectionintegrated microfluidic chip detection platform.Methods: The design of microfluidic chip is mainly composed of two parts: theconcentration gradient generator (CGG) and the cell culture chamber. CGG wasproduced according to the modified methods described by Jeon et al, and constructed adrug screening system for promoting nerve regeneration, with PC12cells as the effectorcells. Polydimethylsiloxane chip was produced as previously described methods,Briefly, a film mask containing the channel design was used in contact photolithographywith SU-8photoresist (Microchem, USA) to generate a negative mold on a cleanedsilicon wafer. Positive replicas with embossed channels were fabricated by moldingPDMS against the SU-8master mold. Then the PDMS replica was cut down and drilledholes using a sharpened needle at inlets and outlets. Finally, the side of the PDMS slabwith embossed channels was irreversibly bonded to a glass slide assisted by oxygenplasma surface treatment. PC12cells cultured on the chip to observe cell growth andproliferation.Results: Constructed by the upstream concentration gradient generator anddownstream of the cell culture unit microfluidic chip.Conclusion: Cell culture Successful in microfluidic chip, that can provide reliabledetection platform for further drug intervention, and protein detection. Part2Role of microfluidic chips in FK506-and nerve growth factor-inducedneurite outgrowth in PC12cellsObjective: Based on a microfluidic chip detection platform and PC12cells, weconstructed a drug screening system for promoting nerve growth, and could fast andaccurately obtain the drug concentration of FK506and NGF on PC12cells neuriteoutgrowth, thus providing the theoretical basis for the treatment of nerve injury.Methods: Firstly screened out the optimal concentrations of FK506and NGF using the microfluidic chip technology, then set8drug compatible concentrations incomparison with their optimal concentration, after PC12cells were incubated for48hours, the NF200expression in PC12cells was detected with an immunocytochemicalmethod, to reflect the neurite outgrowth states. To verify the accuracy of microfluidicchip platform, we also incubated the same batch of PC12cells onto96-well plates withthe same conditions.Results: FK506(200nM) triggered the greatest effects on the neurite outgrowth inPC12cells, and there were significant differences compared with other groups (P <0.05). NGF (50ng/mL) also produces the greatest effects on promoting the neuriteoutgrowth in PC12cells, with significant differences compared with other groups (P <0.05). Combined application of FK506(114.29nM) and NGF (21.43ng/mL) allows thegreatest effects on promoting the neurite outgrowth in PC12cells, with significantdifferences compared with other groups (P <0.05). The two-sample average t-test wasused to compare with96-well culture plate method, and results showed no statisticallysignificant differences between the two methods (P>0.05).Conclusion: The combined application of FK506and NGF has a synergisticeffect in promoting nerve regeneration. Combined application of FK506(114.29nM)and NGF (21.43ng/mL) allows the greatest effects on promoting the neurite outgrowthin PC12cells. Compared with the traditional detection platform, microfluidic chipdetection platform has many advantages analysis faster, less reagent consumption, easyintegration and high-throughput analysis. Part3Combined treatment of FK506and nerve growth factor forspinal cord injury in ratsObjective: To observe whether FK506and NGF has a synergistic effect onpromote the neural regeneration and on the recovery of spinal cord functions followingacute spinal cord injury in rats.Methods:120female rats were randomly divided into five groups, namely controlgroup, FK506treatment group, NGF group, FK506+NGF group, and sham surgery group, with24rats in each group. Models of spinal cord injury were established usingthe modified Allen’s method. At30minutes after injury, rats in each treatment groupwere treated with FK5060.3mg/kg, NGF40ug/kg, FK506+NGF (0.3mg/kg+40ug/kg) via intraperitoneal injection, once a day, for successive one week. The NF200expression in rats with spinal cord injury was determined using immunohistochemicalstaining, NF200mRNA expression levels were observed with reversetranscription-polymerase chain reaction method and the restorations of spinal cordfunctions were assayed with Basso, Beattie and Bresnahan score at3,7,14,21dayspost-injury.Results: At7,14,21days post-injury, the NF200staining average intracellularoptical densities, the optical densities value ratio of NF200mRNA, and the BBB scorewas gradually increased in all treatment groups and the control group, and the treatmentgroups showed significantly higher expression levels than the control group and shamsurgery group (P <0.05)Conclusion: FK506and nerve growth factor have a synergistic effect in thetreatment of spinal cord injury in rats, the combined treatment can effectively promotethe neural regeneration and functional recovery in rats after spinal cord injury.