动物造模,周围神经 z-bo 2020-09-10 354

　　Introduction: The peripheral nervous system (PNS) of mammals maintains a high regenerative capacity, and even adults can achieve long-distance regeneration and axonal function recovery. This opportunity for regeneration is reduced in adult mammals, slowed down, incomplete and/or ineffective peripheral nerve repair. However, the limited progress in understanding the molecular and cellular mechanisms of PNS aging has hindered the development of rational rehabilitation treatment for elderly patients. Therefore, our aim is to discover how aging affects the maintenance and regeneration of peripheral nerves. After trauma, peripheral nerves undergo multiple steps of repair, including wall degeneration, axon regeneration and targeted innervation. The characteristics of Waller’s degeneration are as follows: (A) separation of Schwan cells from related axons, (b) transformation of these Schwan cells into "repaired Schwan cells" phenotype, (c) destruction of the blood nerve barrier, and (D) tissue In macrophages, it flows in and swallows axons and mirin-derived fragments together with (E) "Repair Schwan cells". In the regeneration phase, macrophages support "Schwan cell repair", mediate axon regeneration and re-regulate target tissues. When the inflammatory process disappears and the "swan cell repair" differentiates again, regeneration is complete. Successful peripheral nerve repair requires several different types of neurons, Schwan cells and immune cells. The intrinsic growth potential of neurons does not seem to be affected by aging. This indicates that the defects of older animals are caused by environmental damage, while the efficiency of aging Schwan cells and macrophages to remove debris is low. Two major studies confirmed that the environment for axon regeneration in older animals is defective. The former observed age-dependent differences in Schwann cell behavior and delayed the activation of the repair program. The reduced in vitro feeding of Schwan cells and macrophages indicates that the slow regeneration of nerves in elderly rodents is the reason why Schwan cells and macrophages cannot repair their functions. The purpose of this study is to investigate the inflammatory neural environment that can regenerate intact old nerves. By affecting the repair process of Schwan cells, we proved that changes in the inflammatory nerve microenvironment are factors that affect the maintenance and regeneration of peripheral nerves in the elderly.

　　Peripheral nerve regeneration from age-related injury: Many studies have shown that the regeneration ability of peripheral nerves decreases with age, but its underlying mechanism is still limited. In order to better understand the effects of age-related factors on peripheral nerve regeneration, two C57BL/6J mice of different ages were subjected to sciatic nerve contusion, with an average life span of 24 months. A mouse that is 20 months old is defined as a "big" mouse, and a mouse that is 6 months old is defined as an "adult" mouse. Older mice exhibit typical aging functions, such as whales and fur. As shown by the Semmes-Weinstein monofilament test, sensory recovery is significantly delayed after sciatic nerve contusion. Most sensory recovery may be due to the sprouting of collateral branches, because the saphenous nerves in the foot area are not damaged and the nerves are over tense, leading to the observation of hypersensitivity. Single-frame motion analysis (SFMA) studies the measurement of the recovery of the bottom corner of the mouse as a highly reproducible marker of functional muscles. Aged mice showed a significant delay in the recovery of motor function and a delay in the recovery of leg extension ability-a surrogate indicator of exercise kidney function after peripheral nerve injury in the leg. Experiments show that the functional recovery of elderly mice is delayed, but almost completely recovered after peripheral nerve crush injury. The electrophysiological properties further reflect the difference in functional nerve repair. The proximal and distal ends of the sciatic nerve were stimulated in situ at the compression site, and the combined neural activity potential (CNAP) and nerve conduction velocity (NCV) of the two groups of intact and damaged nerves were evaluated. Four weeks after contusion, the CNAP of aging mice was significantly lower than that of adult mice, and the number of functional regeneration axons was reduced. Nerve damage in old mice showed that NCV slowed down and myelin sheath decreased. To assess nerve regeneration at the structural level, half-thin sections of the intact control and the sciatic nerve were analyzed 4 weeks after the contusion. Damaged nerves in adult mice appear as small myelin axons, similar to myelin axons and almost no macrophages. Older mice have fewer axons, smaller diameters, thinner myelin sheaths and more macrophages. The quantification of myelin thickness relative to axon diameter shows the main difference between the two groups of regenerative nerves, especially for the larger axon diameter. Studies on axon density, average axon diameter and myelin thickness show that there are defects in sciatic nerve regeneration in old mice, and the g ratio does not decrease with age. The immunohistochemical staining results of the sciatic nerve at different time points after injury were similar. Older mice developed delayed Waller degeneration 3 days after nerve injury, and then delayed incomplete myelination. In the 4th and 8th weeks after injury, axon regeneration has little effect on aging. This can be seen on the distal end of the Remyrin sheath in the 4th and 8th weeks after the injury. On the other hand, axonal regeneration can be confirmed in the sciatic nerve of aged mice after injury. The CNAP observed was significantly reduced. This is due to the aforementioned insufficient innervation, and may also be due to age-related changes in neurotrophic factors from soluble targets. Our data show that 4 weeks after nerve injury, the morphological regeneration of peripheral nerves in the elderly is insufficient. We hypothesize that Schwan cell function declines with age, rather than the unique characteristics of axons, which is the reason for the decline in the ability of peripheral nerve repair in the elderly.

　　Injury response of the elderly and changes in the inflammatory microenvironment: Waller’s degeneration is a prerequisite for effective regeneration of injured nerves, including several different cell types, including macrophages and other immune cells, which are effective for regeneration of injured nerve Premise. IBA-1 immunostaining was performed at different times before and after the contusion to identify the macrophages in the sciatic nerve of adults and the elderly. The number of macrophages in the sciatic nerve of aged mice appears to increase, which is not related to injury, and suggests that aged nerves have a chronic inflammatory microenvironment. Compared with adult mice, the number of macrophages in old mice after nerve injury (3 days) was significantly reduced, but the macrophage infiltration was significantly excessive in the later stage. This is consistent with the data shown in the thin-walled section. IBA-1 immunoblotting of the sciatic nerve lysate confirmed this finding, indicating that the accumulation of chronic macrophages in the peripheral nerves of old mice is low, and continuous and sustained damage can cause inflammation. The age-related changes in the inflammatory microenvironment before and after contusion were analyzed, and various cytokines, chemokines and acute-phase proteins in the neurolytic fluid of adult and old mice were screened. Age-dependent changes in cytokine expression levels can be detected in injured and intact nerves. At 3 days after injury, the expression in adult mice increased significantly, but was effectively down-regulated at 8 weeks after injury. Three days after injury, the cytokine activity of aging mice decreased, and after 8 weeks, the up-regulation rate increased. The expression of cytokines in aging mice is delayed but prolonged. In intact nerves, comparison of cytokine profiles confirmed the age-dependent down-regulation of anti-inflammatory cytokines interleukin 4 (IL-4), IL-13 and IL-27, and pro-inflammatory cytokine monospheres. Age-dependent up-regulation of cell chemotactic protein 1 (MCP1) and CC chemokine ligand 11 (CCL11). Anti-inflammatory treatment strategy: Acetylsalicylic acid (ASA) suppresses the natural immune response of mammals and reduces the infiltration of ischial macrophages. In order to test whether inhibiting the high inflammatory response induced by the injury of ASA to elderly mice can improve peripheral nerve regeneration, a 4-week treatment plan was established in two groups of elderly mice. Starting from 3 days after injury, "ASA" animals received low-dose ASA (PBS 10mg/kg) every 2 days. The "vehicle" control animals received only the same amount of PBS. Use SFMA and toe diffusion analysis to monitor the recovery of motor function, and use the Semmes-Weinstein monofilament test to monitor sensory function to test the treatment effect. ASA processing has obvious advantages for all test parameters. Cytological analysis confirmed the inhibitory effect of ASA treatment on the persistent inflammatory response in aging mice after 4 weeks. Cytokines were down-regulated below the undamaged control level, including MCP1 and CCL11, and the effect of ASA on macrophage infiltration was observed 4 weeks after extrusion in the longitudinal section. Macrophages are usually stained with IBA-1, and pro-inflammatory M1 and pro-regenerative M2 macrophages are identified by iNOS and arginase 1. The quantification of staining is for (a) total cell density (stained with dapi), (b) total number of macrophages (iba-1), (c) inflammatory M1 macrophages (iOS) and (d) precursor regenerated M2 macrophages Phage is very important. Will be reduced to. The decrease in inflammation is accompanied by an improvement in remyelination, which is an increase in the myelin protein 0 (MPZ) signal in the tissue section and the formation of the 21.5-kDa subtype of myelin basic protein (MBP) throughout the neurolysis process. Sexual upregulation is manifested as specificity. After ASA treatment, pho-erk 1/2 increased slightly, which also showed the increase in regeneration capacity. In addition, electrophysiological measurements showed that CNAP and NCV increased after ASA-treated elderly mice were compressed for 4 weeks, but this trend did not reach a statistically significant level. .. Our data show that after low-dose ASA treatment, injury-induced inflammation is significantly reduced, highlighting the beneficial effect of anti-inflammatory treatment on peripheral nerve regeneration in aging mice.

　　"CCL11 inhibits Schwan cell myelination in vivo and in vitro: focusing on CCL11 and MCP1 to clarify the relationship between peripheral nerve inflammation and myelin remodeling in the elderly continues. with age. Denervated Schwan cells express MCP1, an effective macrophage attractant. CCL11, also known as eosinophil immune cell chemokine, is secreted by M1 and M2 macrophages. Both cytokines are locally expressed in the sciatic nerve within two days after injury. We have determined the local up-regulation after explant culture, which supports the important role of normal peripheral nerve repair and inflammation. High levels of MCP1 may be the reason for the increased infiltration of macrophages in the old intact sciatic nerve, but the effect of CCL11 on the peripheral nerves of the elderly remains unclear. CCL11 binds to CC chemokine receptor (CCR) types 2, 3, and 5. However, transcriptome analysis has hardly detected the expression of CCR3. CCR3 is the main receptor related to eosinophil attraction, but it can significantly express CCR2 and CCR5. In addition, like the ligand CCL11, CCR5 is also Up. After peripheral nerve injury, Schwan cells and macrophages both express CCR5 and are found to have a significant up-regulation effect. We hypothesized that CCL11 may be directly involved in the regulation of Schwan cell behavior and tested it in a system co-cultured with DRG neurons and Schwan cells. DRG isolated from mouse embryos was cultured in medium for 6 days, and then cultured in myelin medium containing CCL11 or carrier for 8 days. Staining and qpcr are used to assess myelin formation. In the samples treated with CCL11, it was found that staining with ΔβMBP as a myelin sheath marker and neurofilament heavy polypeptide as a neuron marker significantly reduced the myelin sheath of each axon. QPCR analysis showed that in the co-cultures treated with CCl11, the expression of myelin markers mpz and mbp was significantly reduced. Dedifferentiation or proliferation markers did not change from other myelin markers. This indicates the special role of CCL11 in myelination. In order to evaluate the effect of CCL11 on Schwan cell behavior in vivo, adult mice were continuously injected with CCL11 or vehicle (PBS) from 1 week before unilateral sciatic nerve contusion and 4 weeks after injury. I've had enough. At the same time, the regeneration of the external intact nerve and remyelination. No changes in macrophage infiltration behavior were observed after injection of CCL11. However, in mice treated with CCL11, the MPZ signal intensity in the compression zone tended to be less remyelinated. Western blot analysis of myelin basic protein (MBP) showed that in CCL11-treated mice, the expression of myelin remodeling was significantly reduced, indicating a decrease in myelin remodeling. QPCR analysis showed that after sciatic nerve injury, the myelin marker mRNA (Mpz, Mbp, Egr2, Prx) in the CCL11 and vehicle treatment groups decreased significantly. Unlike injured nerves, there were no significant differences between the two groups of intact sciatic nerves. This indicates that CCL11 has a particularly large effect on remyelination.

　　Conclusion: When there is intact chronic CCL11 in the intact nerve, Schwan cells seem to enter a continuous dedifferentiated non-functional repair mode, thus impairing the maintenance of peripheral nerves. In addition, the existence of injury-induced CCL11 will impair Schwan's regenerative cell repair activity and maturity. We have determined that CCL11 is an important age-dependent inflammation-inducing circulatory factor and a promising therapeutic target for improving the maintenance and repair of peripheral nerves in the elderly.