Optimization Of Functional Imaging Approach On MEMRI In Rat Brain For Evaluating Functional Reconstruction Following The NSCs Transplantation

BackgroundThe objective of this topic is to provide an initial basis for magnetic resonance functional track of the neuron-like cells differentiated from neural stem cells(NSCs) after transplantation.NSCs play an important role in the process of nerve injury repair. NSCs iderived from bone marrow stromal cells (BMSCs-D-NSCs) have a wide perspective of clinical application because of rich source, autologous transplantation without immune rejection and ethical problems. Our research team has made positive results in the differentiation and transplantation of BMSCs-D-NSCs. To study the mechanisms of NSCs transplantation for neural tissue repair further, it is necessary to assess the founction of NSCs at some certain point after transplantation.But so far all the technologies including patch-clamp technique of brain slice in vivo have no breakthrough in the functional trace of transplanted NSCs. It is uncertain how the transplanted NSCs reorganize and play related functions in vivo by integrating with the nerve tissue of the host. It is important to identify and assess the function of BMSCs-D-NSCs in vivo for its application in the treatment of CNS injury and repair. The literature search and related results of previous studies indicated that NSCs have neuron-like cell differentiation capabilities in vitro in terms of morphology, neural electrophysiology, neural biochemistry. iPS technology, as the recent development and a fervent concern, indicated that stem cells including NSCs could differentiate into neurons and show some of the related function of neurons. These findings at early stage have provided a favorable basis for us to research the functional assessment of transplanted BMSCs-D-NSCs in vivo. A more effective and direct method is urgently needed to assess the activity of differentiated cells after NSCs transplanted in vivo.Green fluorescent protein (GFP) and 5-bromo-2'-deoxyuridine (BrdU), the markers of NSCs are usually used as the tracing lalels in vivo following the NSCs transplantating. However, both of then can be observed and analyzed only by specimens section, but not trace in vivo. However, the differentiation and functional evaluation of the NSCs in vivo is more important.The survival and migration research of the transplanted stem cells structurely tracked in vivo, have been reported. Especially the transplanted cells marked by superparamagnetic iron oxide (SPIO) were well traced during their repairing myocardial defects in vivo. Our laboratory has achieved positive preliminary results. For example, the transplantation of BMSCs-D-NSCs marked by SPIO could repair CNS damage in vivo. But until now, the functional imtegrated status between the nerve tissure and the transplanted BMSCs-D-NSCs could not be tracked.The functional assessment and tracer research about the integration between the transplantated stem cells and the host tissue in vivo have seldom been reported before. The FDG PET and BOLD MRI methods can only provide information about regional brain activity-changes at the glucose metabolism level. It remains difficult to directly reflect the changes of neural activity, particularly the functional activities of the transplanted NSCs at the cellular level in vivo.Therefore, our experimental team applicated the manganese-enhanced magnetic resonance image (MEMRI) technology to functionally assessment the neuron-like cells differentiated from transplanted NSCs here.The mechanism of activity-induced manganese-enhanced magnetic resonance functional imaging technology is described as below. Manganese ion (Mn2+) could go into the corresponding activated cerebral nuclei or cortex through an open voltage-gated calcium ion (Ca2+) channel while the nerve stimulation occurs because the chemical nature and ionic radius of both Mn2+ and Ca2+ are similar. The voltage-gated calcium channel is the most important factors involved in synaptic activity and related neurological function; therefore we observe the neuronal functional activity using Mn2+, instead of Ca2+, through MRI. We can also observe that Mn2+ could aggregate in the corresponding cerebral nuclei of the sensory system and the cortex by MRI after MnCl2 was administrated systematically and relevant sensory stimulation was given.Mn2+ can quickly enter the active neurons when the nerve function activity takes place while its removal from the cell needs a long period. Thus, Mn2+ continues to increasely aggregate in the active neurons which results in the time extending of nerve stimulation and drug duration. This term cumulative effects of Mn2+ imaging will contribute to record more subtle and transient electrical activity of neurons from tens of millions transplanted cells, which is the advantage of our technology and different from the BOLD fMRI or brain slices patch-clamp technique. Furthermore, Mn2+ imaging applys to catch the characteristic of the less number of neuron-like cells with quantity and functional activity at instantaneous. But the Mn2+-enhanced functional imaging technology is in developing stage, there are still many methodological problems.First, the toxicity of Mn2+, including acute and chronic toxicity, is one of the important factors disturbed functional assessment of neuron-like cells differentiated from the transplanted NSCs. The acute toxicity, affected by the dose and manner of the administration, can not be overlooked especially in Mn2+ -enhanced functional magnetic resonance imaging tests. The status of Ca2+ channel such as activation or is lock depends upon the different doses of Mn2+. The toxic of Mn2+ on myocardium is the main aspects of its acute toxicity. Hence different dosage, administration manner and speed of Mn2+ inhibit cardiac power delivery system at varying degrees. Doses of Mn2+ applied in the past MEMRI tests were relatively high, which was not suitable for the functional evaluation of the neuron-like cells differentiated from transplanted NSCs. Therefore, it is the first and necessary step to reduce the applicated dose of Mn2+, which will be focused in this study. Secondly, T1-weighted sequence of signal intensity is an indicator that reflects the concentration of Mn2+ accumulation in MEMRI. Although past studies have shown that Mn2+ concentration in vivo have a linear relationship with T1 relaxation time, T1-weighted signal intensity was impacted not only by the T1 relaxation time, but also the T2 relaxation time. Therefore, T1-weighted signal intensity have a linear relationship with the concentration of Mn2+ only within a certain range. It is also necessary to measure the concentration of Mn2+ in cortex in order to further support the imaging results. In our research, "inductively coupled plasma mass spectrometry (ICP-MS)" as a kind of chemical assay way was employed, in order to support the data of MEMRI.Thirdly, data processing of MEMRI need to explore further, because there is no uniform method of data processing procedures currently.Overall, functional evaluation in vivo of the neuron-like cells differentiated from transplanted NSCs requires a mature and effective method of MEMRI. The purpose of our topic is to optimize the technique candations of the MEMRI fore evaluating the function of the transplanted NSCs in brain. It is most important to reduce the dose of Mn2+ through improving the method, to establish the chemical detection methods for supportting the imaging results, and to find an appropriate processing method of imaging data. So we try to set up a set of testing program for the functional evaluation of the neuron-like cells differentiated from transplanted NSCs in vivo.PartⅠMEMRI on rat primary cortical neuronsObjective:To explore the feasibility of showing neurons functional activation after labeled with manganese. It is necessary to know the absorptive capacity and imaging characteristics of neurons, the concentration of neurons and Mn2+ administration that could enhance imaging results, and the effect of Mn2+ on neurons in order to evaluating the function of neuron-like cells differentiated from the transplanted NSCs by MEMRI. Preliminary study of MEMRI on rat primary cortical neurons was conduted to understand the above problems, provide experimental basis for functional imaging of the neuron-like cells differentiated from transplantated NSCs in vivo, and select appropriate number of transplanted cells and dose of Mn2+ in the next experiments.Methods:Test 1:Primary cortical neurons, cultured and identified with conventional methods, were divided into control group and MnCl2 intervention group, which was divided into 0.05,0.1,0.2, and 0.5 mmol/L MnCl2 subgroup. The MnCl2 intervention groups were administered with MnCl2 at different concentrations, and the control group was same volume of saline without MnCl2. The effect of MnCl2 on cellular survivial rate at different concentrations administered for 24 hours was assessed by MTT assay. Test 2:Primary cortical neurons were divided into control and, with concentratins of 0.05,0.1,0.2, and 0.5 mmol/L respectively MnCl2 intervention group. Cells in the intervention groups with 0.05,0.1, and 0.2 mmol/L of MnCl2 were simultaneously added with 10μmol/L glutamic acid in order to stimulate the neurons. Mn2+ content and magnetic resonance imaging were measured by inductively coupled plasma mass spectrometry (ICP-MS) at 24 h after the intervention with MnCl2 and glutamic acid. SPSS 13.0 statistical software package was used to analyze the data. Analysis of variance was used to analyze measurement data. LSD was used for group comparison.Results:The primary cortical neurons showed growed well. Test 1:MTT results showed that cellular survival rates in MnCl2 intervention group were significantly lower than those in the control group (Pab=0.000; Pac=0.000; Pad=0.000; Pae=0.000). The number of the surving cells in the 0.2 and 0.5 mmol/L MnCl2 intervention groups were higher than those in the 0.05 and 0.1 mmol/L MnCl2 intervention group (Pbd=0.009; Pbe=0.004; Pcd=0.001; Pce=0.000). Test 2:ICP-MS analysis showed that Mn2+ content in neurons was higher along with the higher concentration of MnCl2 added. Mn2+ content inintervention group with 10μmol/L glutamic acid and 0.5 mmol/L MnCl2 was significantly higher (840ppm) than that (140ppm) in 0.5 mmol/L MnCl2 intervention but glutamic acid-free group. The MRI signal intensity in 0.1,0.2, and 0.5 mmol/L MnCl2 intervention group were significantly enhanced than those in 0.05 mmol/L MnCl2 intervention group and the control group. The signal intensity in T1-weighted sequences increased with concentration of MnCl2 increased, and the signal intensity in adding glutamic acid group higher than that in simple MnCl2 intervention group.Conclusions and implications:1. MEMRI on cells cultured in vitro was established.2. It is possiable to trace the function of neuron-like cells differentiated from NSCs by MEMRI. At least,106/ml of neurons with 0.1mMol/1 of MnCl2 could show the effective MEMRI. These results proved a certain extent viability of MEMRI on neuron-like cells differentiated from transplantated NSCs, and the basis for next experiment.PartⅡ:Low dose of MnCl2 functional magnetic resonance imaging on rat visual cortexObjective:To explore the optional conditions for excellent MEMRI, including the lowest dose of MnCl2 and the best way, with which the time of manganese role can be well enlonged. Recent study revealed that the usual dose of MnCl2 in MEMRI was as 33 mg/kg on rat visual cortex, and its possitive effect had to be showed by board-level analysis. To reduce the dose of manganese and at same time to enhance the imaging effect must be as the important title to be considered and resolved. Past studies have implied that Mn2+ could persistently go into the active zone after the blood-brain barrier (BBB) closed. In this study, we will explore whether or not the migration process of Mn2+ really exists and the total amount of Mn2+ can be reduced through extending the stimulation time after the closure of BBB.Methods:Adult male Wistar rats were randomly divided into visual stimulation, no-stimulation group, semi-stimulation group, and MnCl2 intravenous injection group. After anesthesia by intraperitoneal injection of 36 mg/mL chloral hydrate (360 mg/kg), rats were accepted intubatton in the right external carotid for injection of drugs. Each group was administrated MnCl2 when the BBB opened after the right external carotid artery injection of 30% mannitol. The former 3 groups were injected MnCl2 by the tube in the right external carotid, the last group was injected MnCl2 (5 mg/kg) by tail vein. Then visual stimulation group was placed in the visual stimulation device for 5 hours, no-stimulation group in the darkroom for 5 hours, semi-stimulation group was placed in the visual stimulation device for 2 hours and subsequently displaced to the darkroom for 3 hours, MnCl2 intravenous injection group was in the darkroom for 2 hours and subsequently displaced to visual stimulation device for 3 hours. MRI images and data were acquisited after visual stimulation. Rats were killed and brain was removed for quantitative analysis of manganese content in the visual cortex by ICP-MS. Images were processed using matrix laboratory (MATLB), statistical parametric mapping (SPM), MRIcro softwares and the process includes image registration, image average of one group, image subtraction and regions of interest (ROI) analysis. SPSS13.0 statistical software package was used to analyze the data. Analysis of variance was used to analyze measurement data. LSD was used for group comparison.Results:The average T1-weighted images showed that the right hemispheres of the former 3 groups, following by damage the BBB by injection of 30% mannitol, enhanced the signal and had significantly difference with the left hemisphere. The signal enhancement in the cortex of MnCl2 intravenous injection group was not observed, but a slight increase in double sides of deep brain structures. The signal enhancement was showed at the visual cortex in the visual stimulation group, at the visual and some non-visual cortex (not focus on specific areas of brain function) in semi-stimulation group, and at the extensive cortex (no specific enhanced region) in no-stimulation group. The pixel-based image subtraction showed that major enhanced region related to visual stimulation was visual cortex in the visual stimulation group, and some non-specific enhanced regions (not focus on specific areas of brain function) in addition to the visual cortex in the semi-stimulation group. ROI analysis showed that the standard signal intensity (SSI) at right visual cortex of visual stimulation group was significantly stronger than that of semi-stimulation group (bcp=0.009). At the same time, the SSI in the semi-stimulation group also showed stronger than that in no-stimulation group (abp=0.020). Manganese content analysis in the right visual cortex supported the results of ROI analysis (bcp=0.029; abp=0.040).Conclusions and implications: 1. The higher signal intensity could be showed through applicating MnCl2 at 1/6 of usual concentration.2. A large number of Mn2+ entered brain through open BBB, and Mn+ in different brain region could persistently go into the active zone after the BBB closed.3. Mn2+ could go into not only the target area, but also the brain or cerebrospinal fluid through the open BBB. The migration process of Mn2+ after passing the BBB would help to reduce the interference caused by blood flow dynamics of intravascular injection.4. ICP-MS was applicated to confirm the relationship between imags signal and concentration of Mn2+, which could make up the shortage of non-linear relationship between the signal intensity and the manganese concentration.PartⅢ:Nasal administration of Mn2+ -enhanced functional magnetic resonance imaging on rat visual cortexObjective:To investigate the manganese imaging mehod in brain by detoured rounol the BBB. The method above still possessed some of shortages in which the BBB is the biggest obstacle of Mn2+ to entrance into the brain. Otherwise, the intubatton of external carotid also belonged to the invasive, and the blood clotting was not conducive to the multiple imaging. Previous studies about imaging of nerve conduction pathways have shown that Mn2+ could enter olfactory bulb through an olfactory pathway. While the olfactory bulb belongs to a BBB-free brain area, therefore, nasal administration of Mn2+ was considered used in this test. Mn2+ in the olfactory bulb would distributed to functional activation domain of brain through the neural pathways, and then achieve functional imaging by bypassing the BBB.Methods:Adult male Wistar rats were randomly divided into the intact olfactory bulb group and the olfactory bulbectomy group. After anesthesia by 2% isoflurane, rats were accepted the intubatton in nasal cavity by injection of MnCl2 at a concentration of 3 mol/L, with 5μL in each side of nasal cavity. Rats then accepted double stimulation of both visual and osmia at the same time. The methods employed to analyze the imaging date were as same as the one in PartⅡ. Results:The average T1-weighted images showed that the signal of olfactory bulbs MRI in intact olfactory bulb group were obviously enhanced, in contrast, no signal enhancement of the olfactory bulb MRI was observed in the olfactory bulbectomy group. At the visual cortex of coronal plane, the MRI signal in deep brain structures and visual cortex were enhanced in the intact olfactory bulb group, while only deep brain structures showed the enhanced MRI signal in the olfactory bulbectomy group. The pixel-based image subtraction showed that major enhanced region related to olfactory bulb was visual cortex in the intact olfactory bulb group. ROI analysis showed that the signal intensity of visual cortex in the intact olfactory bulb group was significantly stronger than that in the olfactory bulbectomy group (t=3.973, P=0.003). Manganese content analysis in the visual cortex supported the results of ROI analysis (t=-2.696,P=0.022).Conclusions and implications:1. The functional imaging of rat visual cortex can be gotten in no-invasive by bypassing the BBB.2. Osmia stimulation following the nasal administration of MnCl2 positively plays an important regulating role in functional imaging.Part IV:Functional mapping of rat brain activation following rTMS using activity-induced manganese-dependent contrastObjective:To further confirm the functional imaging artery administratin of MnCl2 after repeatative transcranial magnetic stimulation (rTMS). The former test confirmed that higher signal intensity on target cortical was found using low dose of MnCl2 functional MRI at the circumstance of the activated visual cortex, opened BBB and extended stimulation time. In order to validate the method is also applicable to other activated brain regions and provides a new imaging research way, whole-brain was activated by using rTMS and mapped by MEMRI.Methods:Adult male Wistar rats were randomly divided into three of groups including high-frequency magnetic stimulation, low-frequency magnetic stimulation, and sham stimulation. After anesthesia by intraperitoneal injection of 36 mg/mL chloral hydrate (360 mg/kg), rats were accepted intubatton in the right external carotid for injection of drugs. Each group was administrated with MnCl2 (5mg/kg) when the BBB opened after the right external carotid artery injection of 7mg/kg mannitol (30%). Then rats of the former 2 groups were placed in the certain for rTMS (high-frequency stimulation whth 10 Hz, and low-frequency with 1 Hz). Rats in sham stimulation group were placed in the same fixtures in which the magnitude of the angle between the coil and rat skull was as 90°, in order to make the rat brain far away from the magnetic field, but only provided with the magnetic field noise as same as the high-frequency and low-frequency magnetic stimulation. MRI data were acquisited after administration of MnCl2 and magnetic stimulation for 5 hours. The methods employed to analyze the imaging date were as same as the former test.Results:The average T1-weighted images showed that the right hemispheres of the 3 groups, injected mannitol to damage the BBB, enhanced the MRI signal and had significantly difference with the left hemisphere. The sinal of right cortex at coronal plane was enhanced in the high-frequency magnetic stimulation group. The signal at right side of coronal plane containing motor and sensory cortex was enhanced in the low-frequency magnetic stimulation group, but no specific enhanced region in the sham stimulation group. The pixel-based image subtraction showed that major signal-enhanced regions related to magnetic stimulation were olfactory bulb, the right cortex containing the motor and sensory visual areas at coronal plane, the righte inferior colliculus in the high-frequency magnetic stimulation group, and the later 2 regions were related to magnetic stimulation in the low-frequency magnetic stimulation group. ROI analysis showed that the standard signal intensity in right motor cortex of high-frequency magnetic stimulation group was significantly stronger than that of low-frequency magnetic stimulation group (bcp=0.009). The standard signal intensity in the low-frequency magnetic stimulation group showed stronger than that in the sham-stimulation group (acp=0.000). Manganese content analysis in the motor cortex supported the results of ROI analysis (bcp=0.000; acp=0.007).Conclusions and implications:1. MEMRI refers to not only the visual cortex but also other encephalic regions, which can be applied to rTMS study.2. A new animal model for rTMS study was provided by this experiment.3. Brain activity can be enhanced under high-frequency magnetic stimulation while that was decreased under low-frequency. The wider range of brain active areas can be obtained by high-frequency magnetic stimulation.