| Background:Spinal cord injury is a worldwide medical problem that needs to be solved urgently because of its great harm to neural function,many complications and high disability rate.Secondary injury is an important part of the pathological mechanism of spinal cord injury,which will increase the scope and degree of neural injury,and has attracted the attention of many scholars.Tanshinone IIA is considered to be effective in inhibiting secondary injury and rescuing spinal cord function,but its liposoluble physicochemical property limits its application.Meanwhile,the existence of blood-spinal cord barrier prevents drug molecules from reaching the segment of spinal cord injury quickly and in sufficient quantity.Therefore,it is very important to explore new dosage forms and administration methods to improve the bioavailability of tanshinone IIA in the location of spinal cord injury and promote its neuroprotective effect.In addition,the specific biological mechanism of tanshinone IIA in the intervention of spinal cord injury deserves further research.Objective:In order to improve the administration efficiency and bioavailability of tanshinone IIA,the drug dosage form(sustained-release preparation)and administration method(intrathecal administration)were ameliorated.To observe the effect of intrathecal administration of tanshinone IIA-PLGA sustained-release microspheres on the recovery of neural function in rats with spinal cord injury.At the same time,the possible biological mechanism in this intervention process was discussed based on Notch signaling pathway with the focus on the M1/M2 phenotypic polarization of microglia.Methods:(1)Tanshinone IIA-PLGA sustained-release microspheres were prepared by emulsification electrospray method.The micromorphology was observed by scanning electron microscope,and the drug sustained-release property was understood by in vitro release test,so as to conduct preliminary quality evaluation of the microspheres.(2)Among fifty-four rats,two rats were randomly selected for occipitocervical anatomical observation after cardiac perfusion to explore the operation details of intrathecal injection,two rats to explore the maximum-safe injection volume of single intrathecal injection,and two rats to conduct intrathecal toluidine blue staining experiment to explore the dispersion and distribution of drug solution in the subarachnoid space.The remaining rats were randomly divided into six groups:three groups without spinal cord injury modeling(normal control group,intrathecal injection sham operation group,and intrathecal injection group),and the other three groups with spinal cord injury modeling(spinal cord injury group,spinal cord injury+intrathecal injection sham operation group,and spinal cord injury+intrathecal injection group).The normal control group did not receive any treatment;Intrathecal puncture and injection of normal saline(simulated drug solution or suspension)were not performed in the intrathecal injection sham operation group after exposure of posterior atlanto-occipital membrane;The intrathecal injection group was given intrathecal injection of 200 μL normal saline(speed:100 μL/min)after exposure of the posterior atlanto-occipital membrane and uniform extraction of 100 μL cerebrospinal fluid(speed:200 μL/min);In the spinal cord injury group,NYU spinal cord impactor was used to cause T9 spinal cord contusion(10 g impactor rod dropped at 25 mm height and hit the spinal cord);The spinal cord injury+intrathecal injection sham operation group received the experimental operations of the spinal cord injury group and intrathecal injection sham operation group successively;In the spinal cord injury+intrathecal injection group,the experimental operations of spinal cord injury group and intrathecal injection group were performed successively.The safety of intrathecal injection via the posterior atlanto-occipital interspace was verified by behavioral evaluations and body mass measurement.(3)Seventy-two rats were randomly divided into six groups:sham operation group,model group,sodium tanshinone ⅡA sulfonate intraperitoneal injection group(sodium tanshinoneⅡA sulfonate group),tanshinone IIA-PLGA sustained-release microspheres low-dosage intrathecal injection group(low-dosage group),tanshinone ⅡA-PLGA sustained-release microspheres medium-dosage intrathecal injection group(medium-dosage group),and tanshinone ⅡA-PLGA sustained-release microspheres high-dosage intrathecal injection group(high-dosage group).In the sham operation group,the T9 and T10 laminae were removed and the T9 spinal cord was fully exposed,and the back surgical incision was closed without spinal cord injury modeling.In the remaining group,NYU spinal cord impactor was used to cause acute T9 spinal cord contusion(10 g impactor rod dropped at 25 mm height and hit the spinal cord).After that,the sham operation group and model group were intrathecally injected with normal saline 200 μL via the posterior atlanto-occipital interspace immediately after the back incision was closed;The sodium tanshinone ⅡA sulfonate(20 mg/kg)was intraperitoneally injected every day in sodium tanshinone ⅡA sulfonate group from immediately after operation to 7 days after operation;The low-dosage,medium-dosage,and high-dosage group were intrathecally injected with tanshinone ⅡA-PLGA sustained-release microspheres suspension(mass-volume concentration of microspheres was 100 mg/mL)via the posterior atlanto-occipital interspace immediately after the establishment of spinal cord injury model,and the dosage was as follows:20 μL/100 g in low dosage group,40 μL/100 g in medium dosage group,and 80μL/100 g in high dosage group.The functional recovery of spinal cordin each group was observed by behavioral evaluations and neuroelectrophysiological examination.The HE and Nissl stainings were performed at 1 w and 8 w after operation to observe the pathomorphological changes of spinal cord tissue at the injured segment in each group.(4)Eighty-eight rats were randomly divided into four groups:sham operation group,model group,sodium tanshinone ⅡA sulfonate group,and medium-dosage group.The experimental operations of each group were the same as that of the corresponding group in(3).The existence and distribution of M1/M2 microglia in spinal cord tissue were observed in situ by immunofluorescence double-labeling staining.The expressions of M1 microglia markers(iNOS,CD86)and M2 microglia markers(Argl,CD206)in injured spinal cord were analyzed semi-quantitatively by Western blotting.The expression levels of pro-inflammatory cytokines(TNF-α and IL-1β)and anti-inflammatory cytokines(IL-10 and TGF-β)in injured spinal cord were detected by ELISA.The Western blotting was used to detect the expressions of Notch signaling pathway related proteins(Notchl,Jaggedl,and Hes1)in injured spinal cord to understand the activation/inhibition of this pathway.Results:(1)Under scanning electron microscope,the tanshinone ⅡA-PLGA sustained-release microspheres were regular spheres,with smooth surface and uniform particle size.In vitro release test showed that the whole release process of tanshinone ⅡA-PLGA sustained-release microspheres was relatively stable,and no burst release was observed.The cumulative release rate of the microspheres tended to be stable at 21st day and later,approaching 80%.(2)There were no special signs of central nervous system injury caused by intrathecal injection during and after operation,and no cerebrospinal fluid leakage was observed.After the operation,all the rats were able to raise their heads normally,and there were no abnormal behaviors such as self mutilation,reduced diet,and listlessness.No dysuria and dysporia was observed in the three groups without spinal cord injury modeling;At 1.5 h after intrathecal injection,the observation of gross specimen showed that toluidine blue solution was widely dispersed in the subarachnoid space with the circulation of cerebrospinal fluid,staining the whole spinal cord blue,and toluidine blue concentration was observed at the injured T9 segment;There was no difference in BBB score,inclined plane test result,and hot-plate test result among the three groups without spinal cord injury modeling at the same time point(P>0.05),and there was also no difference among the three groups with spinal cord injury modeling(P>0.05);Although the intrathecal injection via the posterior atlanto-occipital interspace caused a decrease in the body mass of rats in a short period after operation due to surgical trauma and other factors,it did not cause a long-term serious decrease in body mass.(3)The BBB score,inclined plane test result,and hot-plate test result were improved more significantly in the medium-dosage group and high-dosage group than those in the model group,sodium tanshinone ⅡA sulfonate group,or low-dosage group(P<0.05),but there was no significant difference in the above behavioral evaluations between the medium-dosage group and high-dosage group(P>0.05);The P-P amplitude of motor evoked potential in the medium-dosage group and high-dosage group was significantly higher than that in the model group,sodium tanshinone ⅡA sulfonate group,or low-dosage group at 1 w and 8 w after operation(P<0.05),and the latency of somatosensory evoked potential in the medium-dosage group and high-dosage group was significantly lower than that in the model group,sodium tanshinone ⅡA sulfonate group,or low-dosage group at 8 w after operation(P<0.05),but there was no significant difference in the above neuroelectrophysiological examination results between the medium-dosage group and high-dosage group(P>0.05);The HE and Nissl staining results showed that compared with other intervention methods,intrathecal injection of high and medium dosage microspheres reduced nerve tissue edema and neuronal injury,inhibited neuronal death,and maintained the arrangement structure of neural fasciculus in white matter at 1 w after operation,and reduced the atrophy of the whole injured spinal cord,reduced the area of cavity inside the injured spinal cord,and promoted the recovery of injured neurons at 8 w after operation.The pathomorphological score of injured spinal cord in the medium-dosage group and high-dosage group was significantly lower than that in the model group,sodium tanshinone ⅡA sulfonate group,or low-dosage group at 1 w after operation(P<0.05),and the transverse diameter of injured segment in the medium-dosage group and high-dosage group was significantly higher than that in the model group,sodium tanshinone ⅡA sulfonate group,or low-dosage group at 8 w after operation(P<0.05),but there was no significant difference in the above HE staining evaluations between the medium-dosage group and high-dosage group(P>0.05).(4)The distribution of M2 microglia in the injured spinal cord of rats in the medium-dosage group was wider and denser than that in the sodium tanshinone ⅡA sulfonate group or model group,while the distribution of M1 microglia in the injured spinal cord of rats in the medium-dosage group was more limited and sparser than that in the sodium tanshinone ⅡA sulfonate group or model group.At 1 w and 8 w after operation,the average fluorescence intensity of iNOS in the injured spinal cord of rats in the medium-dosage group was significantly lower than that in the sodium tanshinone ⅡA sulfonate group or model group(P<0.05),while the average fluorescence intensity of Argl in the injured spinal cord of rats in the medium-dosage group was significantly higher than that in the sodium tanshinone ⅡA sulfonate group or model group(P<0.05);At 1 w and 8 w after operation,the expressions of iNOS and CD86 in the injured spinal cord of the medium-dosage group were significantly lower than those of the sodium tanshinone ⅡA sulfonate group or model group(P<0.05),while the expressions of Argl and CD206 in the injured spinal cord of the medium-dosage group were significantly higher than those of the sodium tanshinone ⅡA sulfonate group or model group(P<0.05);At 1 w and 8 w after operation,the expression levels of TNF-α and IL-1β in the injured spinal cord of the medium-dosage group were significantly lower than those of the sodium tanshinone ⅡA sulfonate group or model group(P<0.05),while the expression levels of IL-10 and TGF-β in the injured spinal cord of the medium-dosage group were significantly higher than those of the sodium tanshinone ⅡA sulfonate group or model group(P<0.05);At 1 w and 8 w after operation,the expressions of Notchl,Jaggedl,and Hes1 in the injured spinal cord of rats in the medium-dosage group were significantly lower than those in the sodium tanshinone ⅡA sulfonate group or model group(P<0.05).Conclusions:(1)The tanshinone ⅡA-PLGA sustained-release microspheres prepared by emulsification electrospray method have smooth surface,uniform particle size,and good sustained-release property.(2)The intrathecal injection via the posterior atlanto-occipital interspace has strong safety,high reliability,and low risk of complications.For rats with a body mass of 220-250 g,the single safe intrathecal injection volume can reach 200 μL.Furthermore,the intrathecal injection via the posterior atlanto-occipital interspace is suitable for the T9 spinal cord contusion model.(3)Intrathecal administration of tanshinone ⅡA-PLGA sustained-release microspheres can significantly promote the repair of neural injury and the recovery of neural function after spinal cord injury in rats.Meanwhile,the high-dosage or medium-dosage intrathecal administration of microspheres has more obvious neuroprotective effect than the conventional intraperitoneal administration of sodium tanshinone ⅡA sulfonate.It is confirmed that the treatment concept of "sustained-release preparation+intrathecal administration" has certain advantages over the conventional treatment concept in the experiment of tanshinone ⅡA intervenes spinal cord injury in rats.In addition,after comprehensive analysis,we determined that the medium-dosage(microspheres:4 mg/100 g,100 mg/mL microspheres suspension:40μL/100 g)was the relative optimal dosage for intrathecal injection of microspheres.(4)Intrathecal administration of tanshinone ⅡA-PLGA sustained-release microspheres can negatively regulate the M1 phenotype and positively regulate the M2 phenotype of microglia by inhibiting the activation of Notch signaling pathway,so as to reduce the pro-inflammation and neurotoxicity of polarized microglia,enhance its anti-inflammatory and neuroprotective function,and ultimately promote the recovery of neural function in rats with spinal cord injury. |
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