Age-related Myelinopathy In The Somatosensory System And Its Implications In Development Of Elderly Pain

BackgroundThere is the problem of population aging in almost all countries of the world; the rapid growth of the aging population will inevitably bring great challenges for social health and public health services. It‘s unavoidable that a significant proportion of older people are experiencing various types of pain. In elderly nursing home, there is at least one pain problem up to 80% of the elderly, more than 40% of people are experiencing excruciating pain. The aging process itself is also a major risk factor for various types of pain. It‘s obvious that the age-dependent incidence increases whether it is the periphery neuropathic pain, central neuropathic pain, or bone and joint degeneration. But the mechanism is still unclear.At present, most pain studies focus on the age-related changes of the unmyelinated fibers, and the myelinated fibers is not attached enough importance to. Some studies suggest that the large myelinated fibers damage may also cause pain mainly due to disinhibition mechanism, but the small fiber dysfunction is thought to be the main reason for the occurrence of pain generally. Due to the long process of elderly pain research, the complexity of mechanisms involved and the lack of good animal models, so the current research on elderly pain is not deep enough. The myelin sheath and axons have undergone significant degeneration in the course of aging, have these changes played a role in the course of neurobiological process in older pain? If have, what is the way that they influenced the elder pain? Objective 1. Observe the age-related changes in the ultrastructure of rat somatosensory system; 2. Detect the age-related changes in the expression of myelin-associated proteins in therat spinal cord; 3. Detect the activation of microglias and astrocytes in the aging rat spinal cord; 4. Evaluate the age-related changes in the rat somatosensory and the somatic movement,and the influence of age on pain behavior in pain models; 5. Investigate the possible mechanisms of the age-related changes in rat somatosensoryand somatic movement. SignificanceWe explored the possible mechanisms of elderly pain from the following aspects : the age-related changes in the myelin structure of the somatosensory system; the age-related activation of glial cells, and the age-related dynamic changes of pain behaviors and inflammatory factors in normal rats and pain model rats, by using transmission electron microscope technique, immunofluorescence and other morphological methods, combined with molecular biology techniques and behavioral methods. According to our experiments, try to demonstrate the complex biological state of older pain from the perspective of myelin and glial cells. MethodsThe transmission electron microscope technique is applied to observe the age-related changes in the ultrastructure of rat somatosensory system. We detect the age-related changes in the expression of myelin-associated proteins in the rat spinal cord and the activation of microglias and astrocytes in the aging rat spinal cord with the immunological method(immunofluorescence and immunoblotting). The age-related changes in the rat somatosensory and the somatic movement, and the influence of age on pain behavior in pain models are evaluated with the behavioral methods. Finally in order to investigate the possible mechanisms of the age-related changes in the rat somatosensory and somatic movement, we applied the immune markers and the immunoblotting method to detect the expression levels of HMGB1 and its receptors(RAGE) in the different age rats. Results 1. The age-related changes in the ultrastructure of rat somatosensory systemThe ultrastructure of rat somatosensory system presents significant age-related changes. The myelinated fiber structure in the PNM2 rat sciatic nerve and posterior funiculus of spinal cord was very complete and matrix full. The pathological changes occurred in part of the militated fibers in PNM12 rats. However, the lesions of myelinated fibers were more apparent in PNM26 rats, these structural alterations including sclerosis of myelin, myelin lamellae splitting and de-compacted, ballooning of myelin and axons, localized demyelination, even the severe disintegration of myelin, and degeneration of axons. In addition, the matrix components lost significantly and the distance between the fibers increased during the aging period. The age-related pathology changes also occurred in the unmyelinated fibers in sciatic nerve. The unmyelinated fiber structure in the PNM2 rat sciatic nerve is complete, and the schwann cell morphology is normal, and the vacuolar degeneration happened in individual axons of PNM12 rats, but the schwann cells weren‘t affected. In PNM26, the pathological changes intensified, the vacuolar degeneration occurred in the unmyelinated axons and even the schwann cells degraded and died, and occurred the necrosis of whole bundle wrapped with schwann cell. In order to better perform the quantitative analysis of the age-related changes of myelin sheath, we applied the pathologically-classified grades method to determine the quantitative grading. Results show that the lesion proportion of the myelinated fibers and the unmyelinated fibers in the sciatic nerve increased significantly with age. At the same time, the lesion proportion of the myelinated fibers in the posterior funiculus of spinal cord also increased significantly with age. 2. The age-related changes in the expression of myelin-associated proteins in the ratspinal cord.Rat spinal cord myelin-associated proteins showed significant age-related changes. The myelin-associated proteins(O1, CNP, Gal C, MOG, etc.) of PNM2 rats CNS presents a complete circle ring. In the aging period, the myelin-associated protein content decreased significantly, especially MOG, CNP and Gal C, DAPI positive nuclei density also decrease significantly. Meanwhile, the results showed that the NF200 in the Posterior funiculus of the aged rats decreased slightly. When both MBP and NF200 are marked, some nerve fibers exhibited MBP and NF200 double positive in aging period. The dense nuclei accumulation happened round the MBP and NF200 coexist fiber, but DAPI and MBP in the aggregated cells can‘t substantially coexist. It‘s identified subsequently that gathering cells can‘t coexist with GFAP, but can coexist with Iba1, namely microglias gather. When the dorsal root ganglion(DRG) cells are marked as different status, the results show the number of both the DRG large cell and small cell didn‘t change a lot with age. After the skin nerve fibers were marked, the number of nerve fibers in the skin of age rats decreased significantly. Meanwhile, we also examined the expression of the myelin associated proteins in the sciatic nerve. The results suggest that the protein expression of CNP, PMP22 and the MBP in the sciatic nerve decreased significantly with increasing age, and the aging cellular skeleton marker(NF200) expression levels were also reduced significantly. 3. The activation of microglia and astrocyte in the aging rat spinal cordThe age-related activation of microglia and astrocyte occurred in the old rats. Few Iba1 positive cells are found in the spinal cord of the younger rats. However, in aging period, the Iba1 positive cells increased significantly, and the microglia activation was apparent. Compared with the white matter, the microglia activation within the gray matter(dorsal horn) in aged rats became more apparent. The Iba1 positive cell density in the older rats increased by about two times than that of younger rats, and in the dorsal horn, they raised almost four times as much. As the immune cells in CNS, the microglias might release a plenty of proinflammatory cytokines and aggravated the disruption of myelin sheath in aging. Similar to the microglia, astrocyte activation is obvious in aging period, but there is no signnificant difference between the distribution of the grey matter and the white matter. 4. The age-related changes in the rat somatosensory and the somatic movement,and the influence of age on pain behavior in pain modelsWe applied PWMT, PWTL to evaluate the changes in rat somatosensory respectively, and used Rot-Rod to assess the age-related changes in somatic movement function. The results suggested that the mechanical hyperalgesia threshold of the older animals(26 months old) significantly increased and the thermal hyperalgesia latency significantly prolonged. Somatic movement evaluation results are similar to the somatic sensory results. In the aging period, the motor function decreased significantly. Rot-Rod residence time was reduced by several times. But when evaluating the impact of age on pain behavior in different pain models, we found that the duration for PWMT in the elderly pain models was significantly longer than the younger. In aging period,the primary mechanical hyperalgesia became more easily persistent and chronic. 5. The possible mechanisms of the age-related changes in rat somatosensory andsomatic movementBased on the foregoing results, we believe that the substance generated by the disintegration of the nervous system myelin and axon activated microglia and astrocyte, caused the immunological reaction, worsened the original damage, and leaded to a series of pain behavior. Therefore we tested the age-related changes of late inflammatory factors(HMGB1) and its receptors expression. The results suggested that the HMGB1 and RAGE expression in the spinal cord of the older animals were significantly higher. Meanwhile the activation of a great deal of glial cells occurred. Similar to the central nervous system, HMGB1 expression in sciatic nerve is age-related. In aging period, it increased significantly more than three times and the expression of RAGE increased by about 20%. Conclusions 1. The pathological changes of the myelin sheath is age-related, the degree of damagedeepened with the age growth. In the process of aging, the myelin sheath is probablythe most easily affected structure in the nervous system, at the same time, it may bethe main targets of ageing. 2. The expression of the myelin-associated proteins in aging period significantly lowered,and the stability of the myelin layer structure is destroyed. It could be the materialbase of the myelin sheath damaged. 3. Microglias and Astrocytes underwent a dramatic activation in aged rat CNS. Andthere were accumulation of microglials around the aged myelinated fibers. Theactivation of glial cells may induce a series of inflammatory reaction and aggravatethe original injury. 4. The normal mechanical hyperalgesia threshold and thermal hyperalgesia latency weresignificantly prolonged in ageing period. But in pain models, the pain reaction ofolder animals became more likely persistent and chronic. 5. We hypothesized that in the process of aging, the degeneration of myelin sheath andaxon may be accompanied by a great deal of HMGB1 emission that activated themicroglia or satellite cells of the central nervous system and peripheral nervoussystem, caused inflammation, worsened the original damage, and leaded to a series ofpain behavior.