Effects Of Myelinated Afferents In Myofascial Trigger Points Induced Myofascial Pain Syndrome

BackgroundsMyofascial pain syndrome (MPS) is a common clinical pain syndrome, which is associated with series of neuropathic pain. MPS is characterized by myofascial trigger points (MTrPs).MTrPs are irritable spots in tant bands of skeletal muscles, which lead to local twitch response (LTR),referred pain and spontaneous electrical activity (SEA) when being stimulated. And they induce motor dysfunction as well, such as the weakness of referred muscle, and limitation of range of motion.Almost everybody exhibits MTrPs, which may exist in any skeletal muscle over the body. However, the genetic of MTrPs is still mysterious. There are mainly two hypotheses about it. Integrated Trigger Point Hypothesis, as well as Cinderella Hypothesis which supplemented this, indicated that the overload of muscle induced local energy crises, excessive of neuroactive substances and lead to sensitization of peripheral nociceptors and hyperalgesia. Another hypothesis, Neuromuscular Junction Dysfunction Hypothesis, considered MTrPs contained lots of motor or sensory related loci, which were responsible for the sensory and motor dysfunctions of MTrPs.Current researches about MTrPs are mainly clinical ones. Some experiments, especially those invasive ones, are hardly to perform on clinical subjects. Therefore, animal models for MTrPs are necessary. In previous animal studies, myofascial trigger spots (MTrSs, equal to MTrPs in human) were induced by beating or eccentric exercise. These treatments were mainly nociceptive, which might result in central sensitization and injuries of myofibers, and disturb the following experimental results. Considering that, a better animal model for MTrPs in humans was needed.In human studies, MTrPs can be identified according to subjective experience, such as local pain (especially recognizable similar pain).Nevertheless, it is nearly impossible to follow that way in animal studies. A modified location method was necessary.Previous clinical studies indicated MTrPs caused hyperalgesia and allodynia, and iron deposition method revealed more nerve endings in MTrPs regions.It was reported that myelinated afferents mediated electrical induced muscular pain. Besides, ischemic compression of myelinated afferents relieved local and referred pain of MTrPs in human beings. Then we inferred, myelinated afferents might be associated closely with MTrPs.In this research, we applied natural developed MTrSs in rats to better imitate MTrPs in humans. An MTrS was preliminary located by palpation and reconfirmed with EMG. Biopsies of MTrSs and non-MTrSs were carried out. Moreover, we applied modified Cajal pyridine silver staining method to indicate free nerve endings in MTrSs/non-MTrSs, and IHC of NF200 to analyze myelinated afferents in MTrSs/ non-MTrSs specifically.Aims1. Location natural developed MTrSs in biceps femoris muscle of rats.2. Study the morphological changes of MTrSs, and compare them with healthy skeletal muscles.3. Confirm the innervations, especially myelinated afferents at MTrSs.Methods1. Location of natural developed MTrSs/non-MTrSs in biceps femoris muscle of rats Wistar rats (weight 260-270 g, SPF level) was lightly anesthetized and immobilized in a prone position. Hair over the biceps femoris muscle was cleared. Tant band was first identified by palpation. Squeezing along the tant band until there existed LTR, and then an MTrS was preliminary located. EMG was applied to conform MTrSs. When SEA was recorded by active polar in MTrS,and no SEA was recorded by control polar in the healthy tissue, an MTrS were conformed. Non-MTrSs had no SEA or LTR.2. Obtain of MTrSs/non-MTrSs in biceps femoris muscle of rats After identifying MTrSs/non-MTrSs, keep the active polar still, and expose the biceps femoris muscle. Remove the muscle surrounding the active electrode (diameter of 3 mm).3. Biopsy of MTrSs/non-MTrSs in biceps femoris muscle of rats. The blocks of MTrSs/non-MTrSs were fixed in 4% paraformaldehyde overnight followed by dehydrated and paraffin embedding. Then the blocks were sectioned to the thickness of 4 μm. Compare the morphological features of MTrSs and non-MTrSs.4. Modified Cajal pyridine silver staining of nerve endings in MTrSs/non-MTrSs According to modified Cajal pyridine silver staining method, the MTrSs/non-MTrSs blocks were fixed, silver staining, reducted with pyrogallic acid, dehydrated and paraffin embedding.4 μm transverse and longitudinal paraffin sections were obtained and observed to analyze numbers of the dark brown nerve endings among myofibers.5.Immunohistochemistry (IHC) of NF200The blocks of MTrSs/non-MTrSs were fixed in 4% paraformaldehyde overnight followed by dehydrated and paraffin embedding.4μm transverse and longitudinal paraffin sections were obtained. Immunohistochemistry of NF200 was carried out to show the NF200 positive myelinated nerve endings. Analyse numbers of the brown myelinated nerve endings.6. Analysis and statistics of dataNumbers of sliver stained/NF200 positive nerve fibers were shown in Mean±SD. t test was applied to analysis the difference between MTrSs and non-MTrSs. The difference was considered as significant if P<0.05.Results1. By applying this method mentioned above, we successfully confirmed natural developed MTrSs and non-MTrSs in biceps femoris muscle of rats.2. Biopsy of MTrSs/non-MTrSs in biceps femoris muscle of rats:myofibers of MTrSs were swollen, disorderly arranged with more inflammatory cells infiltration, while myofibers of non-MTrSs were neatly arranged with fewer inflammatory cells infiltration.3. Modified Cajal pyridine silver staining. In longitudinal sections, silver stained nerve endings were dark brown and could be found between myofibers; in transverse sections, there were more sliver stained nerve endings in MTrSs (15.97±1.67) than non-MTrSs (11.06±.54). And the differences were significant (P<0.01).4. Immunohistochemistry of NF200. In longitudinal sections, NF200 positive myelinated nerve endings were brown and could be found between myofibers; in transverse sections, more myelinated afferents were found in MTrSs (5.720±1.31) than non-MTrSs (2.930±1.09).And the differences were significant (P<0.01).Conclusions1. Natural developed MTrSs could be identified by palpation associated with EMG2. Myofibers of MTrSs were swollen and disorderly arranged with more inflammatory infiltration.3. There were more nerve endings, especially more myelinated afferents, in MTrSs.4. Associated with previous clinical researches, myelinated afferents might related with MTrSs.BackgroundsCentral sensitization is the overreaction of central nerve system, and it is the common mechanism of neuropathological pain. Spinal cord is plastic and modifiable. Continuous peripheral nociceptive stimuli activate neurons in spinal dorsal horn and leads to rearrangement, such as sprout of nerve fibers and phenotypic switch, which results in increased expression of receptors, and release of some new neurotransmitters.All these changes increase the peripheral nociceptive reception threshold and response intensity. Therefore, central sensitization exists. Expressions of glumate receptors have been wildly used to identify the existence and degree of central sensitization.It was reported that myelinated afferents participate in the central sensitization of inflammation and injury. Besides, some scholars indicated that central sensitization exhibit in pathology of MPS. In Part I of this research, we found there were significantly more myelinated afferents in MTrSs than non-MTrSs. Previous clinical researches indicated myelinated afferents were associated with pain and SEA of MTrSs. Therefore, we inferred that myelinated afferents might take part in central sensitization of MTrPs.In addition, scholars applied horseradish peroxidase (HRP) nerve tracing technique to study the sensory neurons associated with MTrSs in dorsal root ganglion (DRG), and found amount and average size of these neurons were smaller than normal. Nevertheless, until now, no such studies about neurons in spinal dorsal horn have been reported. We first applied cholera toxin B subunit conjugated horseradish peroxidase (CB-HRP), a nerve tracer for myelinated fibers specifically, to study the MTrSs associated interneurones in spinal dorsal horn and motor neurons in spinal ventral horn.Most of the clinical proofs were indirect, and some direct central morphological changes were not easy to obtain. Therefore, in order to study the relations between MTrSs and myelinated afferents further, and get more direct proofs, we employed rats in this study, and tetrodotoxin (TTX) was used to block myelinated afferents of MTrSs. Spinal changes associated with MTrSs as well as the TTX blocked MTrSs were analyzed and compared with normal spinal cord respectively.Aims1. Study the relations between myelinated afferents and central sensitization of MTrSs.2. Study the relations between myelinated afferents and MTrSs related neuronal changes in spinal cord.Methods1. Immunohistochemistry of glumate receptors (mGluRla/mGluR5/NMDAR1). 1) Subgroups:24 Wistar male rats with certain MTrSs were randomly divided into 3 groups:NS/MTrSs/TTX group. Another 8 rats without MTrSs were used as control group (normal group).2) Animal treatments:For rats in NS/TTX group,0.1ml NS/TTX (1μM) was injected into MTrSs every 24 hours, and 6 times in all.24 hours after the last injection, L5 spinal cord, which was most closely related with MTrSs in biceps femoris, was removed and made into frozen sections (8μm). For normal/MTrSs group, no special treatment was given, and frozen sections of L5 spinal cord was obtained as well.3) Immunohistochemistry of glumate receptors (mGluRla/mGluR5/NMDAR1). For each glumate receptor, the IHC was carried out in all 4 groups. And the expressions of each receptor, including locations and percentage of positive neurons, were analyzed.4) Analysis and statistics of data. All data were shown in Mean±SD. A one-way ANOVA followed with Newman-Keuls Multiple Comparison Test was used to analysis the difference between groups. The difference was considered as significant if P<0.05.2.CB-HRP nerve tracing technique to study the MTrSs/non-MTrSs related neurons in spinal cord.1) Subgroups:24 Wistar male rats with certain MTrSs were randomly divided into 3 groups:NS/MTrSs/TTX group. Another 8 rats without MTrSs were used as control group.2) Animal treatments:For rats in NS/TTX group,0.1 ml NS/TTX (1μM) was injected into MTrSs every 24 hours, and 6 times in all.24 hours after the last injection 20μl CB-HRP was injected into MTrSs. For normal/MTrSs group,20 μl CB-HRP was injected into non-MTrSs/MTrSs.3) Preparation of frozen sections.48 hours after the injection of CB-HRP, all rats in 4 groups were deeply anesthetized, and were pre-perfusion with NS, fixative (consisted of paraformaldehyde, glutaraldehyde and phosphate butter) and glutaraldehyde. L5 spinal cord was removed and made into frozen sections (10am).4) Observe the locations of CB-HRP labeled neurons. And according to their locations as well as the relations with pain of MTrSs, amount and average size of these neurons in ventral/dorsal horn were analyzed.5) Analysis and statistics of data:All data were shown in Mean±SD. A one-way ANOVA followed with Newman-Keuls Multiple Comparison Test as hoc test was used to analysis the difference between groups. The difference was considered as significant if P<0.05.Results1.IHC of glumate receptors.1) In results of 4 groups, for the same receptor (mGluRla/mGluR5/NMDAR1), locations of positive neurons were almost the same.MGluRla positive neurons located mainly in deep laminae (Lamina III-IV), mGluR5 positive neurons located mainly in superficial laminae (Lamina I-II); while NMDAR1 positive neurons could be found spread the spinal dorsal horn.2) Percentage of positive neuron according to their location. For the certain location of the 3 receptors, there were significant differences among the four groups (P<0.05): the percentage of normal group was the lowest, that of TTX group was in the middle, and percentage of MTrSs/NS group was the highest; Newman-Keuls Multiple Comparison Test revealed no significant difference between MTrSs and NS groups (P>0.05), and differences between any other comparisons of the 4 groups were significant (P<0.05).2. CB-HRP labeled neurons.1) For all groups, neurons that were labeled into bright blue-green by CB-HRP spread over the spinal cord. And the labeled neurons’ locations in 4 groups were similar: mainly in Laminae Ⅰ, Ⅲ, Ⅳ,Ⅴ (lateral 1/3) and IX of the spinal cord.2) Average diameter of CB-HRP labeled neurons:for neurons in the same location of spinal ventral horn and laminae of dorsal horn, a one-way ANOVA revealed significant differences among the four groups (P<0.05):MTrSs/NS group had the smallest neurons, normal group had largest neurons; and the results of TTX group were just in the middle; post hoc test revealed no significant differences between MTrSs group and NS group (P>0.05), while significant differences exhibited between the other comparisons (P<0.05).3)Numbers of CB-HRP labeled neurons:for neurons located in certain lamina of the spinal dorsal cord, a one-way ANOVA revealed significant differences among the four groups (P<0.05):MTrSs/NS group had the fewest labeled neurons, normal group had the most, while the results of TTX group were just in the middle. For neurons in the same lamina of spinal dorsal horn, post hoc test revealed no significant differences between MTrSs group and NS group (P>0.05), while significant differences exhibited between the other comparisons (P<0.05). For neurons in Lamina IX of ventral horn, there were significant differences among the four groups (P<0.05):most labled neurons existed in normal group and less in the other 3 groups; post hoc test indicated no significant difference between any two of MTrSs, NS, or TTX group (P>0.05), but significant differences exhibited when any of the three groups compared with normal group (P<0.05).Conclusions1. Myelinated afferents were related with central sensitization of MTrSs.2. Myelinated afferents were related with plastic changes of neurons in the spinal ventral/dorsal horn associated with MTrSs.3.Selective block myelinated afferents with TTX could revise these central changes mentioned above.