Retinitis pigmentosa (RP) is a disease that seriously affects vision. It mainly affects rod cells and causes night blindness. At the end of the disease, due to the simultaneous involvement of cone cells, the patient’s central vision and peripheral vision loss are not effective. There is no effective treatment method. However, some studies have found that although the function of photoreceptors is lost in the pathological process of RP, the function of bipolar cells and ganglion cells and the neural connection with the visual center are preserved, which provides a condition of therapeutic application in optogenetics for optogenetics. Optogenetics controls the excitability of neurons by expressing the light-sensitive protein represented by rhodopsin ion channel protein-2 on neurons, and has shown great application prospects in reshaping the photoreceptor function of the retina. The treatment of a type of retinal degenerative disease provides an effective treatment option.
ObjectiveTo explore the light sensitivity and kinetic of the new optogenetics tools Channelrhodopsin-XXM2.0 (XXM2.0) and Channelrhodopsin-PsCatCh2.0 (PsCatCh2.0), and analyze whether they could be used to restore the visual function by optogenetics.MethodsMolecular biology techniques were used to link the gene fragments of XXM2.0 and PsCatCh2.0 to the vector pCIG(c)-msFoxn3 containing ampicillin resistant screening gene and reporter gene to form new plasmid pCIG(c)-msFoxn3-XXM2.0 and pCIG(c)-msFoxn3-PsCatCh2.0. The constructed plasmids were transfected into HEK 293T cells, and light responses were recorded in the whole cell mode with the HEKA patch clamp system. The photocurrent was recorded under three light intensity included 2.7×1016, 4.7×1015, and 6.4×1014 photons/(cm2·s). And then, XXM2.0 and PsCatCh2.0 were stimulated with 2.7×1016 photons/(cm2·s) and fully recovered. The opening and closing time constants were analyzed with Clampfit 10.6 software. At the same light intensity, photocurrents of XXM2.0 and PsCatCh2.0 were recorded by the light pulse stimulating of 2-32 Hz. The current attenuation was analyzed at long intervals of 4000 ms and short intervals of 200 ms after repeated stimulation. Comparisons between groups were performed by independent samples t test.ResultsRestriction endonuclease sites of EcoRⅠ and EcoRⅤ were successfully introduced at XXM2.0 and PsCatCh2.0 sequences. When the digestion was completed, they were ligated by T4 DNA ligase to construct new plasmids pCIG(c)-msFoxn3-XXM2.0 and pCIG (c)-msFoxn3-PsCatCh2.0, and then transfected on HEK 293T cells. The light intensity dependence was showed in XXM2.0 and PsCatCh2.0. The greater light intensity was accompanied by the greater photocurrent. Under the light intensity 6.4×1014 photons/(cm2·s) below the retinal safety threshold, large photocurrent was still generated in XXM2.0 and PsCatCh2.0 with 92.8±142.0 and 13.9±5.6 pA (t=1.24, 1.24; P=0.28, 0.29). The opening time constants of XXM2.0 and PsCatCh2.0 were 23.9±6.7 and 2.4±0.8 ms, and the closing time constants were 5803.0±568.2 and 219.9±25.6 ms. Compared with PsCatCh2.0, the opening and closing time constant of XXM2.0 were both larger than PsCatCh2.0. The differences were statistically significant (t=7.10, 31.60; P=0.00, 0.00). In terms of response frequency, XXM2.0 and PsCatCh2.0 could follow to 32 Hz high-frequency pulsed light stimulation, and all could respond to repeated light stimulation at a long (4000 ms) and a short time (200 ms) interval with the small current decay rate.ConclusionXXM2.0 and PsCatCh2.0 could be activated under light intensity with safety for the retina, and could respond to high frequency (at least 32 Hz) pulsed light stimuli with low current attenuation, which could meet the characteristics of opsins required to restore the visual function by optogenetics.
Diabetes and its complications that seriously threaten the health and life of human, has become a public health problem of global concern. Glycemic control remains a major focus in the treatment and management of patients with diabetes. The traditional lifestyle interventions, drug therapies, and surgeries have benefited many patients with diabetes. However, due to problems such as poor patient compliance, drug side effects, and limited surgical indications, there are still patients who fail to effectively control their blood glucose levels. With the development of bioelectronic medicine, neuromodulation techniques have shown great potential in the field of glycemic control and diabetes intervention with its unique advantages. This paper mainly reviewed the research advances and latest achievements of neuromodulation technologies such as peripheral nerve electrical stimulation, ultrasound neuromodulation, and optogenetics in blood glucose regulation and diabetes intervention, analyzed the existing problems and presented prospects for the future development trend to promote clinical research and application of neuromodulation technologies in the treatment of diabetes.