Xiaohua He, MD, MS, Veronica Dishman, PhD
Palmer College of Chiropractic-Florida,
Port Orange, FL 32129, USA.
OBJECTIVE: This study used a unilateral knee joint immobilization model in adult guinea pigs to test the hypothesis that retrograde degeneration of motor neurons in the spinal cord is the result of attenuation of knee joint activities.
METHODS: A total of 32 adult guinea pigs were used and divided into 8 groups based on the duration of knee joint immobilization. Light microscopic studies of Nissl, nitric oxide synthase immunohistochemistry, horseradish peroxidase, and fast blue were carried out to examine the neurons in the spinal cord. Electron microscopy was also performed to examine the neurons and axons.
RESULTS: After various periods of knee joint immobilization, a variety of features of motor neuronal degeneration were observed. Specific characteristics included gradual increases in the expressions of neuronal nitric oxide synthase and ultrastructural changes in affected motor neurons including reduction of cell organelles, indentation of the nuclear envelop, and small compact clumps of chromatin in the nuclei. Observation of the peripheral nerve (femoral nerve) also revealed demyelination alterations in some axons innervating the muscles of the knee joint. Interestingly, motor neuronal degenerative changes and demyelination were reversible after the knee joint immobilization was removed and knee joint activity was restored. These findings may assist in further development of models for spinal dysfunction such as the chiropractic subluxation complex.
CONCLUSION: We conclude that motor neuronal degeneration in the spinal cord and axons in this study was the result of knee joint immobilization. Increases in motor neuronal nitric oxide-mediated oxidative stress level after reduction of target tissue activity may contribute to the mechanism for degenerative changes in the motor neurons in adult spinal cord of the guinea pig.
From the Full-Text Article:
Knee joint immobilization is a common medical practice used to manage musculoskeletal injuries. However, immobilization of the knee joint for a period generally causes degenerative alterations in many articular tissues. Long-term immobilization of animal knee joints leads to progressive histological changes. These changes include proliferation of connective and synovial tissues, fibrous adhesions at articular surfaces, and cartilage erosion and necrosis with subchondral bone alterations. In addition, immobilization also appears to result in a reduction in mechanical strength of joint structural components, thereby leading to progressive degenration. [1-11] Most of these studies have emphasized the effect of joint immobilization on the articular tissues; however, little attention has been given with regard to the study of the neuroanatomical and neurophysiological changes as the result of joint immobilization.
Previous studies by numerous authors on different animals, including cats, [12, 13] rats,  rabbits,  monkeys,  and guinea pigs,  have shown that the knee joint and muscles acting on the knee joint are richly innervated. Various neural elements have been found in different types of articular tissues. It has been demonstrated that knee joint immobilization affects the number and size of the mechanoreceptors in the articular tissues in both rabbits and rats. [17, 18] Using electromyographic recordings, another study showed a transient reduction in the number of motor units after knee immobilization in human subjects.  All of these studies suggest that, together with the articular tissues, the neural tissue in the knee joint will also be altered after knee joint immobilization. However, to date, there have been no further studies to investigate the effects of knee joint immobilization on the nerves innervating the muscles acting on the knee joint and motor neuronal response in the spinal cord.
The present study was designed to assess the ultrastructural, neurohistological, and neurochemical changes in the spinal motor neurons and nerve fibers innervating the muscles acting on the guinea pig knee joint as a result of knee joint immobilization. Unlike other animal models in which some forms of nerve injuries were involved, [20-22] this model did not introduce any traumatic injuries. The purpose of this study was to investigate the nature of the influence of the peripheral effectors (muscles) on motor neurons in the central nervous system without interruption of physical contact between the two.
To the best of our knowledge, this is the first study to show that knee joint immobilization, which alters the normal movements of the knee joint, results in some degree of degenerative changes in both motor neurons of the spinal cord and their axons innervating muscles acting on the knee joint. Previous studies of knee joint immobilization have shown multiple alterations of the articular tissues, especially the articular cartilage. However, spinal motor neuronal response to knee joint immobilization has not been well studied.
Most of the previous studies on the motor neuronal degenerative changes in the spinal cord have been involved in peripheral nerve lesions, such as nerve avulsion  and spinal nerve transaction.  As a consequence of such lesions, the spinal motor neurons showed chromatolysis, loss of Nissl bodies, and dramatic perikarayal shrinkage, leading to apoptosis.  In the current study, however, no nerve lesions were involved; yet, the data revealed a series of progressive motor neuronal and axonal degenerative changes over periods of knee joint immobilization. These changes consisted of axonal demyelination, up-regulated neuronal NOS-LI, folding of nuclear envelope, and increases in nuclear chromatic density. Despite the presence of these neuronal degenerative changes, the reduction of motor neuronal number and the increase of nuclear DNA fragmentation, signifying cell death, in these motor neurons were not observed.
Interestingly, the neuronal degenerative changes observed were reversible upon removing the joint immobilization. Thus, it appears that knee joint immobilization in this model causes reversible neurodegenerative changes that will not lead to neuronal death. Although the structural and molecular mechanisms for these neuronal degenerative changes after RKJI are not fully understood, a plausible etiology is the deprivation of target-derived neurotrophic factors  and nitric oxide-mediated oxidative stress,  which may play a role in neuronal degeneration.
Deprivation of Target-Derived Neurotrophic Factors
A recent study suggested that the proper development of spinal motor neurons was regulated by the Foxp1 gene, which coordinates the identity and connectivity of motor neurons.  Motor neuronal development and survival are regulated by influences associated with both afferent and target contacts. Target-associated influences are regulated by activity such as neuromuscular transmission or muscle contraction.  Normal muscle activity has been indicated to play an essential role in the refinement of neural connections, alterations in axonal fasciculation, and the expression of cell adhesion/guidance molecules.  More importantly, normal muscle activity increases the expression of several molecules associated with the action of neurotrophins on synaptic function and neurite outgrowth in the motor neurons. One study showed that the levels of phosphorylated protein, total protein, and mRNA for synapsin I were elevated in the spinal cord of voluntary exercised rats.  In the immobilized knee joint, it is conceivable that the muscles acting on the knee joint would be drastically deprived of their physical activities. Although the physical connection between motor neurons and muscles is still intact, the functional connection between motor neurons and muscles is hampered. There are several consequences on muscles and motor neurons that could be caused by knee joint immobilization. First, the muscular atrophy as the result of knee joint immobilization could reduce the number of motor units ; second, long-term immobilization would also decrease the activity of axonal transportation, as shown in this study. It is possible that RKJI influences are several with regard to muscle-derived neurotrophic factors.
Target-derived neurotrophic factors may play several key roles between motor neurons and muscles, such as the interaction between motor neurons and their targets, maintenance of neuronal physiological function, and retention of structural constituent of neurons. Some studies have shown that although target-derived nerve growth factor (NGF) dose not prevent motor neuronal death,  other NGF-related molecules, such as brain-derived neurotrophic factor and neurotrophin , support motor neuronal development.  An earlier study showed that the application of ciliary neurotrophic factor to the cut end of the facial nerve rescued motor neurons of the facial nucleus.  Some recent studies have also suggested that the Schwann cell is also a source of neurotrophic factors that participate in the formation, function, maintenance, and repair of neurons. [34, 35]
The results of the current study appear to be in agreement with the above-mentioned studies. The reduction of FB retrograde labeling neurons in the ipsilateral nerve in this study could be explained as the result of reduced motor units and slower axonal transportation—a sign of decrease in functional connection between targets and motor neurons. It is reasonable to assume that such reductions (number and activity) would also affect the influence of target-derived NGFs on motor neurons, leading to increases in neuronal NOS in the motor neurons and a series of degenerative-like changes.
Nitric Oxide-Mediated Oxidative Stress
One of the major findings of this study is increases in neuronal nitric oxide-mediated oxidative stress as a result of RKJI, as evidenced by elevation of the number of neuronal NOS-positive neurons. Analysis of the time course of appearance of NOS-positive neurons and duration of the RKJI suggests a close relationship. Interestedly, the number of NOS-positive motor neurons was diminished after joint release from immobilization. Therefore, it is certain that the elevation of NOS-positive neurons is the result of immobilization and increases in NOS-LI result in nitric oxide-mediated oxidative stress to those motor neurons. In this study, neuronal nitric oxide-mediated oxidative stress level showed a clear relationship with the duration of the RKJI; the longer the immobilization, the higher the concentration. With the reduction of muscle activities, possible also synaptic contacts, and Schwann cell demyelination as the result of immobilization, the influx of Schwann cell-regulated neurotrophic factors and muscle-derived neurotrophic factors by retrograde axonal transport to the cell body would potentially be reduced. This would lead to nitric oxide-mediated oxidative stress in motor neurons as shown in this study, even without the presence of neuronal lesion.
Increase in motor neuronal nitric oxide-mediated oxidative stress was observed in nerve injury models, such as nerve root avulsion  and nerve transection,  and lead to neuronal death. What differs from the nerve injury models is that this study found no neuronal loss. Yet, some neuronal degenerative-like changes, such as the reduction of cellular organelles, folding of the nuclear envelope, and increases in heterochromatin density, were still observed. The relationship between increases in neuronal nitric oxide-mediated oxidative stress level and neuronal degenerative changes is not clear in this study. However, a growing body of evidence suggests the involvement of neuronal nitric oxide-mediated oxidative stress in neurodegeneration. For example, nitric oxide-mediated oxidative stress after nerve lesions would cause subcellular degenerative changes before neuronal death, including interruption of the integrity of mitochondria, redistribution of rough endoplasmic reticulum, shrunken nuclear envelop, and small compact clumps of chromatin.  Although these findings are not identical, they are similar to the findings of this study.
It is plausible that knee joint immobilization may block the transportation of neurotrophic factors, which in turn induces neurotoxicity resulting in activation of Ca2+-dependent enzymes, including neuronal NOS. These enzymes produce reactive oxygen and nitrogen species, which oxidatively modify nucleic acid, lipid, sugar, and protein, leading to nuclear degenerative changes. Because the physical connection between target and neurons is not interrupted, the neuronal nitric oxide-mediated oxidative stress level may not be high enough to lead to cell death. Long-term effect of knee joint immobilization on spinal motor neuronal degenerative changes should be further studied.
This study clearly showed histological and ultrastructural degenerative changes in the peripheral nerve fibers and the spinal motor neurons after various periods of the knee joint immobilization in the guinea pigs. Such degenerative changes were not associated with neuronal death and were reversible.