Study On The Application Of CaMPARI In A Rat Model Of Trigeminovascular Migraine

Background The pathogenesis of migraine is not entirely clear, trigeminal nerve vascular pain pathway activation is thought to be the most fully revelation in migraine pathogenesis mechanism, this theory believes that in migraine model rats, trigeminal ganglion acts as the beginning of a classic upward pain pathway, the activation of it plays a key role in the process of migraine onset and maintain. However, the mechanism of activation of trigeminal ganglion cells is unclear. The traditional methods had confirmed the activation of trigeminal ganglion cells can trigger an explosive rise of intracellular calcium ions, which characterized by a high degree of cell specificity. But in the process of migraine onset and maintain, the activation of trigeminal ganglion cells is still not known,specifically how to activate, activation sequence, etc. CaMPARI fluorescent protein, a new type of neuron calcium imaging of calcium ion indicator, was published in Science on February, 2015. It was characterized by highly sensitive to intracellular calcium ions and irreversible fluorescent conversion, therefore has enormous potential in investigating the activation of trigeminal ganglion cells in the process of migraine onset and maintain. It has been expected that CaMPARI could be used as a marker for the activation of trigeminal ganglion cells, making the instantaneous activation of neurons realized into permanent fluorescent protein expression. However,for now, CaMPARI has only been proved expression model animals tissue like zebra fish, fruit flies and mice, here the question is whether it is available been transducted into rats trigeminal ganglion cells by adeno-associated virus, thus offer a new method for us to study activation of trigeminal ganglion cells in the migraine rats model? The aim of this study is to answer this question.Aim Whether CaMPARI could be transducted into rat trigeminal ganglion cells by adeno-associated virus.Methods 1 To investigate whether AAV-9-hSyn-GFP could be transducted into the trigeminal ganglion cells of rats.First, using methylene blue to determine the stereotaxic injection coordinates of trigeminal ganglion in 200-250 male SD rats. Second, AAV - hSyn - 2 - GFP, AAV - hSyn -8 - GFP, AAV - hSyn - 9 - GFP three serotype AAV were injected into rats trigeminal ganglion in three different doses, 2, 5 8 uL. Control group were given same amount of saline injection. Last, after 2-6 weeks, all the rats sacrificed, all frozen sections of rats trigeminal ganglion were observed the GFP green fluorescent protein expression under fluorescence microscope, to determine the the serum type and dose of the virus in subsequent experiment.2 The AAV - hSyn - 9 - CaMPARI adeno-associated virus preparation.3 Validation of CaMPARI fluorescent protein expression in rats trigeminal ganglion cells.Healthy male SD rats, 2 to 6 weeks after the stereotaxic injection of 8 uL AAV - hSyn-9 - CaMPARI into rat trigeminal ganglion, frozen sections were conducted fluorescence microscopic imaging. Saline injection group acted as a blank control. Avoiding light in the process and the trigeminal ganglia was put in the kerb's solution, which does not contain calcium.Results Two weeks after injection, CaMPARI fluorescent protein could express in rats trigeminal ganglion cells and the expression could last for 28 days at least.Conclusions CaMPARI could be transducted into rats trigeminal ganglion cells by adeno-associated virus, which provide a new method of investigating the role of trigeminal ganglion cells in the process of migraine.