The Pharmacodynamics And Mechanisms Of BBB-Permeable BDNF Fusion Proteins Against Neuronal Death After Cerebral Ischemic

BACKGROUNDBrain-derived neurotrophic factor (BDNF), a member of neurotrophic factor family, is a samll size protein, which was firstly characterized from pig brain by Barde, a Germany neurobiologist, in1982. BDNF is a secreted protein from brain neuron and glial cells. BDNF plays an important role on certain neurons of the central nervous system, helping to support the survival of the neurons, and encourage the growth and differentiation of new neurons and synapses; under both pathological and physiological state, BDNF can promote the repair of damaged neurons. For example, endogenous BDNF can be compensated through their own adjustment when without external intervention under brain ischemia; the neurons can increase their anti-ischemic capacity by BDNF, promote neuronal repair, regeneration of damaged neurons, and reconstruction of neural structures in vivo and promote the recovery of cognitive function after traumatic brain injury. But the compensatory ability of the organism itself is limited, the duration of compensation is short; when the cerebral ischemia was extension, the expression of endogenous BDNF are gradually decrease, while the nerve cell injury repair processes and growth need to have a long time, therefore, the expression levels of endogenous BDNF and receptor tyrosine kinase receptors (trkB) were not sufficient to combat the cerebral ischemic injury and subsequent neuronal cell death in vivo. A large number of studies have been showned that exogenous BDNF can resistant to nerve injury, promote neuron repair, restoring and maintaining neuronal functions in the central nervous system. Therefore, the application of these exogenous BDNF reach the brain by administration and play a biological function, which would be a potential treatment of ischemic brain injury.However, due to the structural characteristics of the blood-brain barrier, limiting the substances exchanged between the blood and brain tissue, therefore, the exogenous protein development is difficult because exogenous protein does not cross the blood-brain barrier. This is so far, there was so few durg used for the prevention and treatment of nervesystem diseases on the pharmaceutical market. How to solve the BBB drug delivery is the key obstacle in the development of BDNF, it also a problem for us, which are most concerned by the researchers in medicine and related areas.Therefore, there is an urgent need for an effective clinical method, non-invasive method make brain-derived neurotrophic factor cross the blood-brain barrier exerts their biological effects, and to meet all there requirements of prevention and therapeutic effect of BDNF in central neural system disease. Currently, there are numerous of drμg delivery methods for therapeutic proteins, mainly included the following methods of administration (invasive and non-invasive methods): cerebrovascular perfusion or intracerebral injection; liposome-encapsulated drugs; the route of administration of the gene therapy by viral vectors; intravenous administration of monoclonal antibody-drug conjugates, etc. However, these drug delivery systems have many deficiencies:cerebrovascular perfusion or intracerebral injection which is invasive method has greatly no-compliance, which also distribution in small parts area after the drug is administration in this way; using viral vectors with BDNF gene delivered into the brain is a good carrier, but is not suitable for the clinical treatment of acute ischemic brain damage, becase the protect and repair of damaged neurons should in a very short period of time by BDNF, in addition, viral vectors delivers that csuse an immune response on human immune system; liposome-encapsulated drugs can increase the lipid solubility of the drug, but the uptake of liposomes into the brain is relatively nonspecific and it also increases the possibility of adverse medication reactions(side effects) and increased costs; the intravenous administration of monoclonal antibody-drug conjugates that can be make BDNF cross the blood-brain barrier get into the brain and play biological effect, but handing of the drugs in this way is cumbersome, the short half-life and high cost of ntibody-drug conjugates have limited its widespread use. So, how to find an effective, non-invasive method of administration of BDNF get into the brain and play its biological effect which is our main major concerns or noted problems that need solving.The TAT protein derived from human immunodeficiency virus which apparently could translocate across the membrane into cells which provides the platform for delivery BDNF into brain. Protein transduction domain (PTD) is less than20amino acids, a basic amino acid-rich region, with positive charge peptide and associated with transduction protein fragments, can cross most of the cell membrane. It can transport of almost all macromolecular into mammalian cells, which without the need for special environment. Penetrating peptide research in the last decades has made great progress in the filed of cancer and immuno-therapy.The preliminary studies was performed in our lab, our lab partner has prepared a brain-derived neurotrophic factor fusion protein (BDNF-PTD) by techniques of genetic engineering. Researchs demonstrate that the BDNF-PTD can pass through the blood-brain barrier and perform its biolobical effects. On this basis, this paper studied the pharmacodynamic and mechanism of BDNF-PTD in the animal models of cerebral ischemia by middle cerebral artery occlusion.OBJECTIVE1、To investigate the application of modified suture in Preparation of rat model of focal cerebral ischemia and to evaluate the stability, repeatability, success rate; Determine the effect of nylon suture coated with paraffin for intraluminal transient middle cerebral artery occlusion. Meanwhile, the study was also to investigate the effect of the two ischemia reperfusion methoda on neurological deficit, infarct size, and blood-brain barrier permeability outcomes.2、To investigate the effect of different dose of BDNF-PTD on the rats dorsal root ganglion neurons; to verify the BDNF-PTD whether or not have the same biological activity and efficacy with the norml BDNF.3、To investigate the neuroprotection on the rat neuron damage induced by cerebral ischemia/reperfusion injury by different doses of BDNF-PTD, and this study was to examine the effects on neurological deficit, infarct area by different doses of BDNF-PTD.4、To investigate the mechanisms underlying the neuroprotective effects of BDNF-PTD in ischemia/reperfusion rat model.METHODS1、Preparation of focal cerebral ischemia and optimization of the Preparation experimentalPreparation of rat model of focal cerebral ischemia by middle cerebral artery occlusion via normal nylon suture and modified nylon suture (Preparation of paraffin-coated nylon filament:Nylon filament (diameter,0.26mm) was cut into70mm long under the microscope. The nylon filament was inserted into polyethylene tubing (Inner Diameter=0.28mm) at the length of8mm, the tubing was filled with melting paraffin for10second, then the tubing was removed after the paraffin coagulated. To maintain consistency, it is recommended that one person prepare all of paraffin-coated nylon filament.). Use of neurological Deficit Scores, TTC staining and Evans blue staining to investigate the effect of MCA occlusion by normal nylon suture and modified nylon suture on neurological deficit, infarct size, blood-brain barrier permeability, meanwhile, investigate the effect of different ischemia/reperfusion injury method on cerebral ischemia model neurological deficit, infarct size, blood-brain barrier permeability, then determine what type of model manner was used in the following experimental.2、The effect of different dose of BDNF-PTD on the rats dorsal root ganglion neuronsPrimary rat dorsal root ganglion neurons were placed at96-well plates, move the plates into the37℃,5%CO2incubator culture, then divided into two groups by randomized after48h, the control group:Primary culture of DRG neurons cultured48h, and then added50μl sterile double distilled deionized water and incubated for48h which was as normal controls; experimental groups:Primary DRG neurons were cultured for48h, then added BDNF-PTD at10ng/ml,50ng/ml,80ng/ml,100ng/ml, BDNF(50ng/ml) and incubated for48h. The morphological features of cell were observed under inverted microscope and photographed. The axonal growth area was shown by percentages in each group (the experimental group compared with negative control group), the axon length values are expressed in μm. All measurement data are expressed as mean±SD, the data was then analyzed by SPSS13.0statistical analysis software.3、The pharmacodynamics of neuroprotective effects against brain neuron death after ischemic/reperfusion injury by BDNF-PTDThe experimental animals were divided into four groups randomly including sham group, vehicle group, the blank model group, treatment group (5Oμg,100μg). The treatment group was administration via intraperitoneal injection of BDNF-PTD50μg,100μg before the middle cerebral artery occlusion was performed in rats and at1h after MCA occlusion and rats were decapitated24hours after stroke modeling. Then the rat brains were quickly removed and were frozen at-30℃for lOmin, dissected into5small coronal slices (2mm thick) and immediately incubated in2%2,3,5-triphenyltetrazolium chloride (TTC) at37℃for10minutes as described as previously reported. The TTC stained viable brain tissue was dark red, whereas infracted tissue was unstained. After TTC staining, the slices were fixed in4%Paraformaldehyde. The border between infarcted and noninfarcted tissue was outlined and analysis with the Image J software, and the area of infarction was measured by subtracting the area of the nonischemic ipsilateral hemisphere from that of the contralateral side. Infarct area were calculated and expressed as a percentage of infarct area to total hemispheric area for each slice.4、The mechanisms of neuroprotective effects against brain neuron death after ischemic/reperfusion injury by BDNF-PTDThe experimental animals were divided into three groups randomly including sham group, the blank model group, and treatment group. The treatment group was administration BDNF-PTD50μg via intraperitoneal injection at1hour after MCA occlusion. And rats were decapitated24hours after stroke modeling, the brain tissue quickly placed in pre-cooled0.9%saline and total proteins were extracted from rat brains using total protein extracted kit, this process are being carried out on ice. Then western blot method used to detect the brain proteins levels of P-Erk1/2, Erk1/2, PI3K and internal control GAPDH protein. We use Gel-Pro professional image software to document and store our western blot data. RESULTS1、Preparation of focal cerebral ischemia and optimization of the Preparation experimentalPreparation of rat model of focal cerebral ischemia by middle cerebral artery occlusion via normal nylon suture (uMCAO) and modified nylon suture (cMCAO). In cMCAO group, there was no rat died after modeling and no rat suffered intracranial hemorrhage, the success rate of model is100%(n=20). While in the uMCAO group (Preparation of focal cerebral ischemia model by normal suture), there were two rats died of intracranial hemorrhage and edema in10hours after modeling, and seven rats were excluded from the success rate account as these rats were found intracranial hemorrhage when sacrificed at12hours, even though these rats showed neurological deficit. The success rate of uMCAO group in this study is55.0%(n=20). Neurologic examinations were performed at6hours after MCA occlusion. Neurological evaluation demonstrated the neurological score of cMCAO group was3.5±0.7(n=20) versus uMCAO group was2.63±0.67(n=20). The cMCAO group had worse neurological deficits than group uMCAO, there was a statistically significant difference between the two groups (P=0.01).Rats were sacrificed12hours after permanent MCAO and assessed the infarct areas with TTC staining. The result showed, in the uMCAO group,11rats brain had infarcts areas (two death, and the brain of seven rats suffered intracranial hemorrhage without showing TTC staining), whereas in the cMCAO group, all rats had infarcts (n=20). The cMCAO group had larger infarct areas than uMCAO group (24.28%±11.31%vs11.57%±5.56%, respectively). The mean of infarct areas was209.97%larger compared with uMCAO group, and there was a statistically significant difference between the two groups (P=0.015). The neurological score examinations were performed at6hours after MCA occlusion. Neurological evaluation demonstrated the neurological score was3.17±0.389in the Group C and3.0±0.426in the group W respectively. there was no statistically difference between the two groups (F=1.000, P=0.328), although the neurological score was higher in group C compared with group W. Rats were sacrificed24hours after MCAO and assessed the infarct areas with TTC staining. There was no statistically different in the infarct area in the group C compared with the group W (21.09±6.53%vs.16.9±4.16%, respectively, P=0.917by ANOVA), althoμgh the mean of infarct areas was124.79%larger in group C compared with group W, there was no statistically difference between the two groups (F=3.5, P=0.075).2、The effect of different dose of BDNF-PTD on the rats dorsal root ganglion neuronsThis part of the experiment shows that in the treatment group, BDNF-PTD could significantly promote the DRG transplantation growth compared with the negative control group and positive group (BDNF) by in vitro co-culture with DRG. This reaserchs show that the neurite length and outgrowth of the DRG transplants was significantly enhanced in co-culture with BDNF-PTD.In the treatment group, there were significant differences in each group (F=31.697, P=0.000). The axon length of DRG, there were significant differences in50ng/ml,80ng/ml,100ng/ml BDNF-PTD and50ng/ml BDNFcompared with lOng/ml (P=0.001), but there were no significant differences in50ng/ml,80ng/ml,100ng/ml BDNF-PTD and50ng/ml BDNF-PTD (P>0.05).3、The pharmacodynamics of neuroprotective effects against brain neuron death after ischemic injury by BDNF-PTDIn the experiment of Pre-BDNF-PTD group, neurologic examinations were performed at6hours after MCA occlusion. Neurological evaluation demonstrated the neurological score of MCAO group was3.67±0.58(n=3), Vehicle group was3.33±0.58(n=3), pre-50μg group was2.33±0.48(n=24), pre-100μg group was2.08±0.65(n=24)。there was statistically significant difference in the groups (F=9.914, P=0.000), there was a statistically significant difference between pre-BDNF-PTD (50μg,100μg) and MCAO group(P=0.000and P=0.000,respectively). In the experiment of Post-BDNF-PTD group, neurologic examinations were performed at6hours after MCA occlusion, Neurological evaluation demonstrated the neurological score of MCAO group was3.33±0.58(n=3), Vehicle group was3.33±0.58(n=3), post-50μg group was2.5±0.78(n=24), post-100μg group was2.33±0.48(n=24)。 there was a statistically significant difference in the groups (F=3.883, P=0.014), there was statistically significant difference between post-BDNF-PTD(50μg,100μg) and MCAO group(P=0.039, P=0.014, respectively)In the experiment of Pre-BDNF-PTD group, rats were sacrificed24hours after MCAO and to assess the infarct size with TTC staining. The result showed, MCAO group, Vehicle group had larger infarct areas than Pre-BDNF-PTD group (50μg,100μg)(P<0.05). there was statistically significant difference between50μg and100μg post-BDNF-PTD (P=0.000)In the experiment of Post-BDNF-PTD group, there was statistically significant difference in the groups (F=12.433, P=0.000), there was statistically significant difference between post-BDNF-PTD(50μg,100μg) and MCAO or Vehicle group(P=0.000, P=0.000, respectively), but there was no statistically difference between the50μg and100μg post-BDNF-PTD(P=0.467).4、The mechanisms of neuroprotective effects against brain neuron death after ischemic injury by BDNF-PTDThe results of densitometric scanning of the PI3K/GAPDH ratio analysis:western blot analysis revealed a strong up-reguation of PI3K/GAPDH levels in BDNF-PTD-IH group compared with sham rats(P=0.005), MCAO-IH (P=0.006)and MCAO-NIH(P=0.002), The difference is statistically significant in the groups (P<0.01). There was also have a statistically significant difference between BDNF-PTD-IH and NIH(P=0.019). In addition, the PI3K/GAPDH levels also increase in the BDNF-PTD-NIH compared with sham rats(P=0.002), MCAO-IH (P=0.002)and MCAO-NIH(P=0.000).The densitometric analysis of the pERKl/2/ERK1/2ratio:western blot analysis revealed a strong up-reguation of pERKl/2/ERK1/2levels in BDNF-PTD-IH group when compared with sham rats(P=0.000), MCAO-IH (P=0.000)and MCAO-NIH(P=0.000), The difference is statistically significant in the groups (P<0.01). In addition, the pERKl/2/ERK1/2levels also increase in the BDNF-PTD-NIH compared with sham rats(P=0.017), MCAO-IH (P=0.020)and MCAO-NIH(P=0.017), but there was also hvae no statistically significant difference between BDNF-PTD-IH and NIH(P=0.957).CONCLUSION1、We conclude that the paraffin-coated nylon filament for intraluminal permanent middle cerebral artery occlusion provide better occlusion than the traditional uncoated nylon filament in the aspects of model success rate, unify infarct area and neurological defects, by virtue of thickness, blunt tip and smooth surface. In additional, These two different intravascular reperfusion methods have its advantage respectively, from the aspect of clinical, the reperfusion blood restoration from CCA in group C is under similar Pathophysiological condition, while not in group W; from the aspect of experimental model operation process, the operation style or process of ischemia/reperfusion experimental stroke model is sampler in group W than in group C, this experiment operation style of experiment is more easier in group W. In conclusion, investigated the effect of different intravascular reperfusion style on ischemia/reperfusion model can improve our understanding of the physiopathology of this experimental stroke model.2、BDNF-PTD fusion protein can promote the neurite outgrowth of DRG Explants in a dose-dependent manner, there is a statistically significant difference between BDNF-PTD and negative control. This result showed BDNF-PTD have the same biological activity and efficacy as the BDNF.3、The results showed that MCAO group, Vehicle group had larger infarct areas than Pre-BDNF-PTD group (50μg,100μg) and post-BDNF-PTD group (50μg,100μg), there was a statistically significant difference decrease in Pre-BDNF-PTD group. Neurological evaluation demonstrated the neurological score of treatment (BDNF-PTD) group was lower than MCAO and Vehicle group. The experiment showed that the BDNF-PTD can across the blood-brain barrier and play a biological effect (improves the ability of neurons against ischemia, protect neurons and improve neurological function). The animal experiments of BDNF-PTD build up a good basis for preclinical research and will serve as a basis for planning future clinical trials.4、The research results show that BDNF-PTD can across the blood-brain barrier and exerts its biological effects, which can offer protection against neuronal damage after cerebral/reperfusion ischemia, promote the survival and development of neurons in vivo. The mechanisms of neuroprotective effect against ischemia/reperfusion-induced brain injury by BDNF-PTD was confirmed that the BDNF-PTD can activates extracellular signal-regulated kinase (ERK) and increased the phosphatidylinositol3-kinase (PI3K) levels, which served as a mechanism of BDNF-PTD exerts its biological effects.