Neuroprotection In The Rat Middle Cerebral Artery Occlusion Model After Intraventricular Administration Of Collagen-binding BDNF Targeting The Ventricular Ependyma

Backround and ObjectiveCerebral ischemic stroke is one of the most common diseases affecting human health. It has a high mortality rate and many clinically difficult sequelae such as hemiplegia, numbness, dysphasia and cognitive deficits.Neurotrophic factors are important for the treatment of cerebral ischemia. One of the most widely distributed neurotrophic factors in the central nervous system is BDNF, which may promote neural regeneration and angiogenesis, and decrease apoptosis and infarct volume. Although there is an upregulation of BDNF expression after cerebral traumatic events, the amount of endogenous BDNF expressed is not sufficient to promote recovery. Therefore, exogenous supplementation of BDNF has high potential for the clinical treatment of stroke.Because BDNF is poorly transported through the blood-brain barrier (BBB), the efficacy of intravenous administration is not clear and it is difficult to control the amount of BDNF delivered. Intraventricular injection is a more effective method for administration of BNDF. Owning to its short half-life and rapid diffusion to cerebrospinal fluid, multi-injections of BDNF or use of an osmotic minipump are currently necessary to maintain local concentrations in the brain, leading to increased wound complications and the risk of infection. Hence, development of a sustained single dose BDNF delivery system is crucial.We have previously demonstrated that BDNF fused with a collagen-binding domain (CBD) specifically binds to collagen. We also found that collagen is abundant in the ventricular ependyma of the brain. Therefore, the collagen of the ventricular ependyma of brain may provide a binding target for CBD-BDNF. Moreover, the subventricular zone (SVZ) is the main region for the generation of new neurons. We proposed that CBD-BDNF would act as an ingenious natural slow release system that would directly stimulate the cell proliferation of the SVZ and exert a therapeutic effect for a longer time. In this study, our collagen-targeting therapeutic strategy was tested in the rat middle cerebral artery occlusion model (MCAO) and the therapeutic effects were estimated by molecular imaging technology, immunohistochemistry and ethology score. MethodsBased on rats MCAO model, The study includes two parts:1. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery of adult rats. At8h after the permanent MCAO surgery, the infarct size become stable. Animals were randomly divided into three groups: NAT-BDNF-grafted, CBD-BDNF-grafted, and medium-grafted groups. Each rat received10ul (0.5nmol) NAT-BDNF,10ul (0.5nmol) CBD-BDNF or10ul PBSinjection slowly for1min into the right lateral ventricle by a stereotactic frame respectively.24rats (n=6, each group of each time point) were executed by cervical dislocation at3h or12h after injection of NAT-BDNF or CBD-BDNF. The infarcted hemisphere was removed and frozen immediately in liquid nitrogen. Then proteins were extracted for western blotting analysis. The Behavioral tests, immunohistochemistry and Micro-PET,SPECT were performed in each group at2weeks after MCAO.2. A stable and permanent acute cerebral ischemia model with unilateral middle cerebral occlusion was established in SD rats and evaluated by micro-SPECT/CT, TTC staining and HE staining within6h of stroke onset once an hour. An independent sample t-test was used to determine the statistical differences between SPECT imaging and TTC staining. A one-way ANOVA was used to analyze multiple comparison procedures.Results1. The distribution of collagen in the ventricular ependyma and choroid plexus of the brain was tested by immunohistochemistry and western blot analysis.2. At both3h and12h after injection, there was significantly more CBD-BDNF remaining in the infarcted hemisphere compared to NAT-BDNF3. Significant functional improvement were observed in the CBD-BDNF treatment group starting at one week after MCAO. The modified neurological severity score of CBD-BDNF group seem to be lower than the control group which demonstrate the better neurological recovery. Administration of CBD-BDNF increased the number of Ki-67-positive cells in the ischemic ipsilateral SVZ compared with the medium control.There are more microvessels in the cortical peri-infarct zones of BDNF group. The number of NeuN immunoreactive mature neurons was also higher in the BDNF group compared to the control groupat the2weeks postischemia. SPECT also showed a significant reduction of the radial rarefaction and defect area in the CBD-BDNF group.18FDG-PET examination displayed a significant reduction of the radial rarefaction and defect area in the CBD-BDNF group.4. In the CBD-BDNF treated group, the number of apoptotic neural cells and the expression of Bax were lower, while the expression of Bcl-2significantly increased5. The infarction zone obtained from SPECT were concordant with the infarction volume from TTC staining for (P>0.05). There was a positive correlation between SPECT low volume infusion areas and the pink areas by TTC staining. The infarct size tends to be constant3h after the occlusion. At the time points of3h,4h,5h,6h there was no significant difference in infarct size.ConclusionCBD-BDNF bound to the collagen of the ventricular ependyma and exerted therapeutic effects more efficiently than NAT-BDNF. In the infarcted hemisphere, CBD-BDNF remained at a higher concentration than NAT-BDNF and stimulated cell proliferation in the SVZ. In the MCAO model, CBD-BDNF promoted cell proliferation, improved perfusion, reduced cell loss, decreased apoptosis, and improved functional recovery in the rat, suggesting that CBD-BDNF may provide an exciting potential treatment for stroke.SPECT as a rapid, accurate, non-invasive method can evaluate the brain haemodynamics of cerebral ischemia animal model in living state, and may have important clinical value for the ultra-acute cerebral infarction for evaluation and decision making.