Neuromuscular damage and repair after dry needling in mice

Abstract

Objective. Some dry needling treatments involve repetitive and rapid needle insertions into myofascial trigger points. This type of treatment causes muscle injury and can also damage nerve fibers. The aim of this study is to determine the injury caused by 15 repetitive punctures in the muscle and the intramuscular nerves in healthy mouse muscle and its ulterior regeneration. Methods. We repeatedly needled the levator auris longus muscle of mice, and then the muscles were processed with immunohistochemistry, methylene blue, and electron microscopy techniques. Results. Three hours after the dry needling procedure, the muscle fibers showed some signs of an inflammatory response, which progressed to greater intensity 24 hours after the procedure. Some inflammatory cells could still be seen when the muscle regeneration was almost complete seven days after the treatment. One day after the treatment, some changes in the distribution of receptors could be observed in the denervated postsynaptic component. Reinnervation was complete by the third day after the dry needling procedure. We also saw very fine axonal branches reinnervating all the postsynaptic components and some residual sprouts the same day. Conclusion. Repeated dry needling punctures in muscle do not perturb the different stages of muscle regeneration and reinnervation.

Figures

Figure 1

Experimental design and sampling. (a)(i) Levator auris longus (LAL) muscle in which repeated punctures are being made with a 0.16 mm thick needle. (a)(ii) Two LAL muscles obtained from the same mouse. (b) Techniques performed and experimental design. After DN, the animals were sacrificed on days one (three hours), two, three, five, and seven. After sacrifice, the muscles were removed, and histological techniques were performed as shown in the figure. In order to minimize the number of animals sacrificed and expenses, the days of extraction and the techniques used were chosen based on a preliminary pilot study.

Figure 2

DN injury in muscle fibers. (a) DN injury in connective tissue covering the LAL muscle for the first three hours. Sometimes, the extremely superficial punctures affected only the connective tissue. At three hours, we can see an incipient inflammatory reaction. Initial magnification: 200x. (b) DN injury in the LAL muscle during the first three hours. Incipient inflammatory reaction at the edges of the lesion can be seen. Initial magnification: 100x. (c) The inflammatory reaction is already complete after 24 hours. Initial magnification: 100x. (d) The inflammatory reaction coexists with the initial stages of regeneration after three days. The area without cellularity is smaller than that seen in images (b) and (c). Initial magnification: 400x. The four images have been stained with methylene blue.

Figure 3

Inflammatory reaction. The figure shows healthy muscle fibers (a) and the inflammatory reaction caused by the needle at the third day after treatment (b). Methylene blue stain. Scale bar: 100 µm.

Figure 4

Myoblast proliferation. When the inflammatory cells remove the debris of necrotic muscle fibers, satellite cells are activated and become myoblasts which initiate mitotic proliferation for several hours. Inflammatory reaction, myoblast cells, and mitotic proliferation are coexisting at the same time. Sample was obtained on day three after treatment. Methylene blue stain. Initial magnification: 1000x.

Figure 5

Muscle regeneration. (a) Myotubes. On the fifth day after puncture, myotubes involved in myofibrillar synthesis can be viewed. Note that there are many areas with newly synthesized contractile apparatus. Some myoblasts have not merged to myotube cells and will become satellite cells in the next days. (b) Young muscular fibers. Seven days after puncture, the DN, signs of degeneration and necrosis are less evident than on day five, and some residual inflammatory cells can be seen. As on the fifth day, some myoblasts attached to the young muscle fibers start regressing to satellite cells. Note that the cytoplasm of the muscle fibers is filled by the contractile apparatus. Some myonuclei definitely remain centralized. Methylene blue stain. Sample included in Spurr. Semithin of 1 µm. Initial magnification: 1000x.

Figure 6

Distal nerve damage by DN. Neurofilament (axon) has been labeled with fluorescein (green) and postsynaptic receptors with α-bungarotoxin rhodaminated (red). (a) Intramuscular nerve shows some axons with fragmented neurofilament. Nerve section due to DN is out of the field, and it cannot be observed in this image. Initial magnification: 600x. (b) The circle shows an example of normal endplate: an axon branched covering postsynaptic component perfectly with compact and defined edges. Inside the box, an example of a recently reinnervated endplate is shown: very thin axon and a dispersed postsynaptic component. Initial magnification: 200x. In (b′), this unstructured component is shown in detail. Initial magnification: 600x. (c) Several days after completion of reinnervation, axonal regrowth can still be seen as in this example: a branch finished in axonal growth cone that runs on the postsynaptic component. Initial magnification: 400x.

Figure 7

Nerve degeneration and glial participation. Transmission electron microscopy (left) of the first 24 hours after nerve injury accompanied by explanatory diagrams (right). (a) Cross-section of degenerating axon surrounded by a Schwann cell that has lost myelin sheath and is ready for phagocytosis. Initial magnification: 5000x. (b) The terminal Schwann cell completely surrounds the axon terminal and phagocytose. Finally, Schwann cell separates the axon and postsynaptic component. Initial magnification: 5000x.