Prevention of disuse bone loss by using implantable micro-electrical stimulators (IMES)

Abstract

Osteoporosis is characterized by extensive decline in bone mass and deteriorated bone micro-architecture leading to increased risk of fracture. Primary osteoporosis refers to osteoporotic conditions associated with aging and decreased gonadal function while secondary osteoporosis is caused by other health problems. Disuse is one of the reasons inducing bone loss resulting in secondary osteoporosis and has been shown to be a regional phenomenon in the areas with tremendous decreases in weight bearing, like the lower limbs. Long term bed rest, spinal cord injuries (SCI), and other brain neurologic conditions leading to substantial reduction in ground force reaction and muscle contraction in the lower limbs as well as exposure to microgravity can lead to disuse osteoporosis. Disuse osteoporosis can be the result of an accelerated rate of bone resorption and slower bone formation, which are associated with the activities of osteoclasts and osteoblasts respectively. Although mechanical stimulation in the form of physical exercise has been reported to prevent bone loss effectively in both humans and animals, patients with physical disabilities and the elderly have difficulty performing exercises. Thus, a safe, easily applicable, and effective preventive treatment for prevention of disuse osteoporosis is needed. The application of electrical stimulation on treating various physiological disorders has been widely reported. Former studies demonstrated that electrical stimulation could increase muscle strength and endurance, and reversed a certain level of osteoporosis in bones stressed by the stimulation in patients who had suffered from spinal cord injuries. However, previous findings on physiological effects of electrical stimulation on bone loss prevention were not consistent. Calcitonin gene-related peptide (CGRP) is a neural peptide secreted by sensory neurons. Previous reports have suggested that CGRP might serve as a local regulator of bone cell function and probably affects bone metabolism via the nervous route as well as via an autocrine loop. Interestingly, CGRP secretion from the dorsal horn could be stimulated by electrical stimulation of a dorsal root. Hence, electrical stimulation of a sensory nerve at its dorsal root in order to trigger CGRP secretion would be a possible treatment for preventing bone loss. Thus, the aim of this study is to investigate the impact of electrical stimulation at dorsal root ganglia on disuse osteoporosis and the underlying mechanism. Sixty male Sprague Dawley rats (male, 3-month-old, 400420 g) were randomly allocated into six equal groups of 10 rats each. The animals were subjected to different treatments according to the following treatment groups: (1) cage control (CC); (2) hindlimb unloaded (HU); and, (3) hindlimb unloaded with electrical stimulation (ES), in which animals were implanted with the implantable micro-electrical stimulators (IMES) before the treatment period. In addition, the effects of treatments were evaluated at two different time points respectively at 2 weeks or 6 weeks. In the ES group, electrical stimuli (treatment parameters: 90μs, 150Hz, 61.3μA and 0.25V) with rectangular waveform and constant amplitude were generated by an implantable micro-electrical stimulator (IMES) and applied directly to the right dorsal root ganglion (DRG). After the treatment period, DRG tissues and tibias were harvested. The expression of CGRP at DRG as well as proximal tibial metaphysis was evaluated by immunohistochemistry whereas the activities of osteoclasts and osteoblasts in proximal tibial metaphysis were indicated by Tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) staining respectively. The change in bone density was estimated by pre- and post-treatment peripheral quantitative computed tomography (pQCT) measurement whereas the bone micro-architecture was evaluated by micro-computed tomography (μCT). In addition, the biomechanical properties of tibias were measured by 3-point bending. Immunohistochemistry results showed that reduced expression of CGRP+ neurons in DRG and proximal tibial metaphysis was observed in HU group whereas direct electrical stimulation at DRG via IMES was able to enhance the expression of CGRP+ neurons in DRG and this enhancement was expected to be associated with the increased expression of CGRP+ neurons at proximal metaphysis of unloaded tibias. The results of TRAP and ALP staining suggested that enhanced osteoclastic activities and suppressed osteoblastic activities would be induced upon unloading since significantly higher density of TRAP+ cells but a lower proportion of ALP active area were observed in the HU group compared to the CC group for both 2-week and 6-week samples. However, no difference in TRAP and ALP was found between CC and ES groups suggesting that suppressed osteoclastic activity and enhanced osteoblastic activity were induced by the electrical stimulation treatment. Further, significant decline in bone mineral density (BMD) and bone mineral content (BMC) as well as deteriorated bone micro-structure were observed in proximal metaphysis of unloaded tibias in HU group as compared to CC after both 2-week and 6-week treatment. However, no significant difference in BMD and BMC as well as bone micro-structural parameters existed between ES and CC at week 2. In addition, after 6 weeks of treatment, no significant difference in BMD and BMC existed between ES and CC as well. However, significant deteriorated bone micro-structure was observed in ES compared to CC after 6 weeks, suggested that electrical stimulation at DRG would reduce decline in bone density by altering the activities of osteoclasts and osteoblasts but the micro-architecture of trabecular bone was possibly regulated by alternative mechanism such as hormonal control. In diaphysis, no significant difference in BMD and BMC as well as micro-structural parameters was found in diaphysis among all groups at week 2. At week 6, no difference in micro-structural parameters in diaphysis was observed between CC and HU but total volume and bone volume of ES were significantly lower than those of CC. The results of pQCT and μCT suggested that electrical stimulation would effectively reduce decline in BMD and BMC as well as preserved bone micro-architecture. Taking the significantly lower averaged body weight of ES animals at week 6 into consideration, the results of μCT would likely be influenced by other physiological or psychological factors. Furthermore, the results suggested that the trabecular compartment was more sensitive to the changes in mechanical loading and electrical stimulation treatment. Based on the results of the current study, direct electrical stimulation at DRG by an implantable device preserves bone mineral density and bone micro-architecture in unloaded tibia by enhancing expression of CGRP+ neurons in DRG and proximal metaphysis. The enhancement in CGRP+ neurons, in turn, suppresses activity of osteoclasts but enhances activity of osteoblasts.