The Experimental Study Of FK506 Treating Acute Spinal Cord Injury

Acute spinal cord injury(ASCI) was a kind of serious damage of nervous system,it brought huge burden to the individuals,the families and the society.The morbidity of ASCI in our country was higher than those of the developed countries.So,it was important to treat ASCI so that to recover or reduce the damage of nervous system.However,the treatment of ASCI was still a difficult problem.ASCI can be divided into primary ASCI and secondary ASCI.Primary ASCI was a mechanical damage.The degree of damage were determined when damage happened.Primary ASCI was non-reversible,and no effective method can be used to treat it;Secondary ASCI happened after the primary ASCI,it was reversible and could be controlled.Secondary ASCI determined the results of ASCI,so the key to treat ASCI was the treatment of secondary ASCI.Now the strategies of treating ASCI were protecting neurons and preventing neurons' apoptosis;promoting the growth of the neurites.Some drugs can influence and interfere the process of secondary damage.The drugs were divided into two groups:Western medicine and Chinese medicine.FK506 named tacrolimus belonged to macrolides antibiotics with strong immunosuppressive action.Recent research showed that FK506 had both immunosuppressive action and neuroprotection,neurotrophy.The mechanism of neuroprotection associated with inhibiting calcineurin(CaN).As a CaN inhibitor,one of the main side effect was neurotoxity.It showed that the notable relativity existed between the incidence rate of neurotoxity and the concentration of FK506.Therefore observing the growth of dorsal root ganglion with different concentration of FK506 to find the best drug's concentration can avoide the neurotoxity and provide a theoretical basis to treat ASCI.FK506 had great side-effect for its easily penetrating blood-brain barrier and low effective plasma concentration.Neurotoxity is one of the main side-effect of FK506.When treating ASCI with FK506,how to achieve the therapeutic effect and reduce the toxic side effect at the same time is the problem faced.One of the ways solved the problem was adjusting the delivery method and speed of FK506.Drug-membrane used locally can provide higher concentration of drugs in the region, sustain-release can reduce the wave of the plasm concentration of FK506. The experiment that using Chitosan sustained-release FK506 incorporated membrane to treat ASCI was carry out to observe the recovery of spinal cord and research the possibility of this method.Ginseng,Salvia miltiorrhiza,Yam,Radix Paeoniae Alba and other drugs composed Zi-Bu-Pi-Yin recipe.Recent research showed that Zi-Bu-Pi-Yin recipe had neuroprotection.But whether the neuroprotection and promoting neurite existed,when Zi-Bu-Pi-Yin recipe combined used with FK506,were unknown.By observing the growth of neurons treated with the two drugs and combined use,the two drugs' neuroprotection and the possibility of combined used to treat the ASCI were researched.Part 1Effects of different concentrations of FK506 on the growth of the new-born rat's dorsal root ganglia neuronObjective:To find the best concentration of FK506 suitable to the growth of the dorsal root ganglia neuron(DRGn)Method:The dorsal root ganglia(DRG) of eight newborn SD rats (within 24 hours) were collected and the epineurium of the DRG was separated.After dissected into about 1mm~3 size,the DRG were digested by 0.25%trypsin.Then DRGn were purified and cultured.Cultured after 96 hours,the neuron was divided into 100pmol/L FK506(Group B),1nmol/L FK506(Group C) and 10nmol/L FK506(Group D),the blank-control group (Group A) was established at the same time.Keep on culturing the DRGn for 48 hours,examine the growth of neurons by NF200 immunofluore-scence, the vitality of the DRGn by MTT method and the expression of GAP-43's mRNA by RT-PCR method.Results:The immunofluorescence results showed that the growth of neurons of Group C were better than Group A,B,D,the growth of neurons of Group B were better than those of Group A,the growth of neurons of Group D were lower than Group A;the OD value of MTT and the expression of GAP-43's mRNA of group B were higher than those of Group A with no statistical significance(P>0.01),the OD value of MTT and the expression of GAP-43's mRNA of group C were significantly better than those of Group A and B(P<0.01),the OD value of MTT and the expression of GAP-43's mRNA of group D were lower than those of Group A(P<0.01).Conclusion:The concentration of FK506(1nmol/L) was the best concentration which having neurotrophy and neuroprotection on the growth of DRGn;100pmol/LFK506 also had neurotrophy and neuroprotection on the growth of DRGn;but 10nmol/L FK506 showed inhibitory action on the neurons.Part 2The experiment of using Chitosan sustained-release FK506 incorporated membrane to treat ASCIObjective:To observe the effects of Chitosan sustained-release FK506 incorporated membrane on the neuroregeneration of the rats after acute spinal cord injury.Method:Allen's method was used to make actue spinal cord injury of the rats.The rats were divided into four groups:sham-operation group(group A,n=20):only cut off vertebral lamina.Damage group(group B,n=20):after cut off vertebral lamina,only damage spinal cord.Chitosan membrane group without drug(group C,n=20):after damage the spinal cord,the chitosan membrane without drug were put up on the damaged spinal cord. Chitosan membrane group with drug(group D,n=20):after damage the spinal cord,the chitosan membrane with FK506 were put up on the damaged spinal cord.BBB judgement were used to observe the rats locomotive function.At the same time,HE staining was used to observe the spinal cord histopathology;immunofluorescence were used to judge the intensity of NF200.Result:BBB judgement showed that group A was better than group B,group C and group D(P<0.05).There was no statistical difference between group B and group C(P>0.05).Group D was significantly better than group B and group C(P<0.05);The histopathology of spinal cord showed that many neurons died and many cavities formed after spinal cord injuried,the number of neurons died and cavities formed of Group D were lower than those of Group B and C;immunofluorescence analysis of NF200 showed that group A was significantly better than group B,C and D(P<0.05).There was no statistical difference between the results of group B and group C (P>0.05).The results of group D was better than group B and group C (P<0.05)Conclusion:Chitosan sustained-release FK506 incorporated membrane used local application can protect the framework of neuron and promote the growth of the neurite,promote the recovery of the spinal cord injury. Part 3The effects of Zi-Bu-Pi-Yin recipe,Tacrolimus(FK506) and combined use on the growth of the dorsal root ganglia neuron of newborn ratsObjective:To observe the effects of Zi-Bu-Pi-Yin recipe,Tacrolimus (FK506) and combined use on the growth of the dorsal root ganglia neuron (DRGn) of newborn rats,discuss the effect of the two drugs on the growth of neuron.Method:The dorsal root ganglia(DRG) of eight newborn SD rats (within 24 hours) were collected and the epineurium of the DRG was separated.After dissected into about 1mm~3 size,the DRG were digested by 0.25%trypsin.Then DRGn were purified and cultured.Cultured after 96 hours,the DRGn were separated into:blank-control group(group A),blankserum control group(group B),Zi-Bu-Pi-Yin recipe group(1%)(group C), FK506 group(1nmol/1)(group D) and combined use group(group E).Keep on culturing the DRGn for 48 hours,examinethe growth of neurons by NF200 immunofluorescence,the vitality of the DRGn by MTT method and examine the expression of GAP-43's mRNA by RT-PCR method.Result:The immunofluorescence results showed that the growth of neurons of Group C,D and E were better than those of Group A and B,the growth of neurons of Group E were lower than Group C and D;There was no statistical difference in the OD value of MTT and the expression of GAP-43's mRNA between group B and group A(P>0.05),the OD value of MTT and the expression of GAP-43's mRNA of group C,D and E were significantly better than those of group A(P<0.05);The OD value of MTT and the expression of GAP-43's mRNA of group C and D were significantly better than those of group B(P<0.05).There was no statistical difference between group E and group B(P>0.05);Compared with group C,group D and E had no significant difference(P>0.05),significant difference was observed between group E and D(P<0.05).Conclusion:1%Zi-Bu-Pi-Yin recipe had neuroprotection and neuroregeneration on the DRGn of newborn rats cultured in vitro;1nmol/l FK506 had neuroprotection and neuroregeneration on the DRGn of newborn rats cultured in vitro;the effect was weakened when1%Zi-Bu-Pi-Yin recipe combined ueded with 1nmol/l FK506.From the above,we conclude that:1.Proving the neuroprotection and neurotrophy of FK506,suggesting that the effects of FK506 were protecting neurons and promoting neurites when treating ASCI.2.Different influence were found on the growth of dorsal root ganglion with different concentration of FK506.When the concentration of FK506 was 1nmol/L,the growth of neurons were significantly better(P<0.01) When the concentration of FK506 was 10nmol/L,the drug inhibit the growth of neurons.3.Chitosan sustained-release FK506 incorporated membrane can reduce the dose of using FK506 and the plasma congcentration.This method can gain good results with low possibility of neurotoxity.Chitosan sustained-release FK506 incorporated membrane using locally can protect neuroframe and promote the neurites so that treating the ASCI of rats.4.Both Zi-Bu-Pi-Yin recipe and FK506 had neuroprotection and neurotrophy,but when the two drugs combined used,the effects were weakened.5.How to use Fk506 including dilivery methods and speed when treat ASCI and how to prevent the neurotoxity of FK506 still need to be investigated.