The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice.
The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.
Skeletal muscle denervation, due to traumatic peripheral nerve injury, disease or pharmacologic intervention, results in the immediate loss of muscle voluntary contractile function. Muscle concomitantly begins to atrophy and this atrophy is reversible if timely, good-quality reinnervation occurs1,2. In the absence of reinnervation, myofiber atrophy progresses, and irreversible biologic changes in the muscle occur with muscle fibrosis and myofiber death. Here we demonstrate the tibial nerve transection model, a model of denervation-induced skeletal muscle atrophy and fibrosis, in mice. This model enables scientists to study the physiologic, cellular and molecular biologic mechanisms that underlie muscle atrophy in vivo in the gastrocnemius and soleus muscles. While historically used predominantly in rats, more recent application of this model to knockout and transgenic mouse lines specifically, allows investigators to assess the role of their particular protein(s) of interest in the induction, development and maintenance, or alternatively the resolution of, muscle atrophy and fibrosis in vivo.
The tibial nerve is a mixed motor-sensory peripheral nerve in the rodent hindlimb, and is one of the three terminal branches of the sciatic nerve. Transection of the tibial nerve denervates the gastrocnemius, soleus and plantaris muscles (and the three small deep flexor muscles of the foot including tibialis posterior, flexor digitorum longus and flexor hallicus longus), and is a well standardized and validated model in rats3,4. The gastrocnemius and soleus muscles can be easily dissected at serial time points post tibial nerve transection, fixed and processed for assessment of muscle histology and muscle fiber morphometrics, or flash frozen for extraction of muscle RNA and protein for the purpose of studying, for example, the cellular signaling networks regulating muscle atrophy. The gastrocnemius muscle is a mixed fiber type muscle (type I and type II, although predominantly type II) and the soleus muscle is composed of a large proportion of type I fibers, thereby providing both fast and slow twitch muscle for assessment5,6. The tibial nerve transection model is suitable for studying the process of denervation-induced muscle atrophy in both the short term (days)7 and long term (weeks to months)4,8.
In contrast to the sciatic nerve transection model (a second model of denervation-induced muscle atrophy commonly used in rodents), tibial nerve transection induces less morbidity in the animal, making it a more attractive model. Transection of the sciatic nerve denervates all the muscles of the leg (below the knee) and foot, impairing the animal's ability to ambulate2, whereas transection of the tibial nerve leaves the peroneal and sural nerve branches of the sciatic nerve intact, thus preserving their target muscles and sensory territories. The mouse is unable to plantar flex or invert the foot, but is able to ambulate easily and the weight bears equally on both hind limbs, thereby significantly diminishing the morbidity of the model. Gait analysis studies evaluating walking patterns have been performed in rats following tibial and sciatic nerve injuries and demonstrate that footprint and weight bearing is better preserved with tibial injury9,10. In addition, in the tibial nerve transection model, the peroneal nerve can be mobilized at a later time point and transferred as a source of delayed reinnervation, if the study design requires3. In contrast, delayed reinnervation in the sciatic nerve transection model necessitates the use of a nerve graft to the sciatic nerve deficit, very significantly increasing the technical difficulty of the model and limiting its use to skilled surgeons.
While the tibial nerve transection model requires familiarity of the operator with sterile operative technique in animal surgery, both the tibial nerve and calf muscles it innervates are easily accessible and identifiable for manipulation, so that individuals who are not surgeons, or highly experienced with animal surgery, can readily master this model.
Prior to using this model, investigators must have received approval for the surgical protocol from their institution's animal use governing body. The model is approved by the Research Ethics Board, Hamilton Health Sciences Corporation, McMaster University (AUP # 10-04-24) and is carried out in strict accordance with the recommendations of the Canadian Council on Animal Care.
1. Mouse Preparation
2. Operative Procedure
3. Post Operative Care
4. Denervated Gastrocnemius and Soleus Muscle Harvest
Tibial nerve transection denervates the gastrocnemius, soleus and plantaris muscles of the calf. Here we assess the development of atrophy in the gastrocnemius muscle, as a representative muscle. Gastrocnemius muscle was harvested from 2-3 months old C57Black 6 mice (Jackson Laboratories) denervated for 1, 2, or 4 weeks. Muscle weights progressively decrease (Figure 1), as does the cross-sectional area of type II fast twitch muscle fibers (Figure 2), over time. The gastrocnemius is a mixed fiber type muscle (type I and type II), but denervation induces a fiber type switch from type I to type II fibers11, and as a result an adequate number of type I fibers may not be available for measurement and robust statistical analysis.
Figure 1. Denervated gastrocnemius muscle demonstrates progressive atrophy. C57Black 6 mice underwent transection of the right tibial nerve. Gastrocnemius muscle was harvested from the denervated (right) and contralateral control (left) hindlimbs at 1, 2, or 4 weeks following nerve transection. Gastrocnemius muscles were weighed, and the weight of the denervated muscle is expressed as a ratio of the contralateral control innervated muscle. Denervation induces a progressive loss of muscle mass.
Figure 2. Denervated gastrocnemius muscle demonstrates progressive decrease in myofiber cross section area (A) Denervated and control gastrocnemius muscles were formalin fixed, cut on cross section at the muscle mid-section and immunostained for anti-skeletal muscle myosin, fast twitch isoform (My-32, Sigma, 1:500 dilution) followed by biotinylated secondary antibody and streptavidin-HRP/DAB as described7. Hematoxylin was used as a counterstain. Fast twitch type II fibers stain brown and slow twitch type I fibers stain light purple. The cross-sectional area (CSA) of the fibers was measured using ImageJ software (Bethesda, NIH) as described7,12. The fast twitch type II fibers demonstrate progressive atrophy. Too few type I fibers are present in denervated gastrocnemius to permit a statistically valid evaluation of fiber size. (n = 6 to 9 mice/group. A minimum of 200 myofibers were measured per muscle by a reviewer blinded to operative phenotype. Data are presented as the mean +/- SD. Scale bar equals 100 μm). Click here to view larger figure.
The tibial nerve transection model of denervation-induced skeletal muscle atrophy is a commonly employed and well validated model in rats. We have adapted this model for use in mice, which allows the investigator to take advantage of the existence of genetically engineered mice and study the process of muscle atrophy in vivo in the absence of proteins crucial to the regulation of muscle mass7,8. The gastrocnemius and soleus muscles, both denervated in this model, can be easily and rapidly dissected with minimal handling, thus providing excellent quality mRNA and protein for subsequent molecular analyses. Similarly, because of the size of the muscles, they can be split, providing tissue from the same animal for concomitant histologic and morphometric analyses. If hindlimb functional assessment is required, walking track analysis can be serially performed. The feet are dipped in ink and the mouse is walked through a enclosure with paper on the bottom. Characteristics of the prints can be reliably measured and scored to indicate the extent of neuromuscular disability and gait compromise, since footprint characteristics reflect the functional muscle groups13,14. While originally developed and validated in rat13, walking track analysis can also be performed in mice15.
Tibial nerve transection is generally extremely well tolerated by the mice. Only a single dose of analgesic is necessary in the immediate postoperative period. With the use of proper sterile technique, soft tissue infection is rare. While tibial nerve transection does induce sensory paraesthesia on the plantar aspect of the foot, in our experience C57black6 and knockout or transgenic mice derived on this line do not tend to auto-mutilate. However, the mice must be inspected daily for signs of auto-mutilation, heel pressure ulcers, as well as point of care endpoints. While we have negligible mortality with the model, we find that approximately 2-5% of mice must be euthanized due to self-mediated injury to, or pressure ulcers developing on, the operated hind limb. The use of soft bedding post-operatively is crucial to ensure the animal's comfort and helps to prevent the development of pressure ulcers on the operated side. Sciatic nerve ligation as well as the SNI model of ligation (where the tibial and common peroneal branches of the sciatic are ligated, but the sural is left intact) serve as models of neuropathic pain16,17. Thus, allodynia and thermal hyperalgesia could occur in the foot in our model as well, but we have not seen overt pain behavior in the mice with normal daily activity on soft bedding.
The tibial nerve of only one hindlimb is transected and since the mice weight bear almost equally on both hindlimbs, the musculature from the contralateral un-operated limb can be used as an internal control within each animal7-10. This is not necessarily the case in the sciatic transection model, where more significant abnormalities of gait can induce a hypertrophic response in the contralateral limb muscle. In the tibial nerve transection model, we typically use the gastrocnemius and soleus muscle from the un-operated limb as our control muscle7,8. If the investigator chooses to use separate animals from which to harvest control muscle, then sham surgery should be performed. Sham surgery would consist of the administration of anesthesia, splitting of the skin to expose the tibial nerve, but no transection. Skin would simply be closed following nerve exposure.
In some peripheral nerve transection models, errant reinnervation from the proximal stump to the target muscle contaminates the planned denervation. In this model, securing the proximal end of the transected tibial nerve to the superficial surface of the biceps femoris muscle, thus closing the muscle interface, inhibits the errant reinnervation. As such, it is a critical and essential step in the model. Errant reinnervation is rare in this model.
Similarly, careful handling of the nerve during surgery is essential. The sural and peroneal nerve branches must be gently separated from the tibial nerve prior to tibial transection, and not crushed or stretched in the process. Rough handling of these nerves will compromise their function, partially denervating other hindlimb musculature. If this occurs the animal's gait will be differentially influenced compared to those mice undergoing sole tibial nerve transection, and the variable muscle loading may contaminate the experimental results. Similarly, care must be taken when dissecting the denervated musculature. Muscle should be handled by the tendon, and not grasped directly to avoid crush artifact that will affect histology, muscle fiber morphometric analyses and possibly gene expression.
We typically employ this model in mice 20-24 g (2-3 months of age), as the animal is mature and the sciatic and tibial nerves are both of adequate size to be easily handled. The surgery can be performed on younger, smaller animals if desired, but the limiting factor here will be the prowess of the operating surgeon. This may be an issue if the investigator is interested in studying the satellite cell response in denervated muscle. The satellite cell regenerative potential diminishes in older, compared to younger, animals18 and therefore younger animals may be required in the experimental design, presenting a technical challenge for a less experienced operator.
The tibial nerve transection model can be adapted from simply a model of denervation-induced muscle atrophy, to one of delayed muscle reinnervation (>4 weeks), if desired and if experienced surgical operators are available1,3. Following a period of denervation specified by the investigator, the mouse can be reoperated and the peroneal nerve mobilized to reinnervate the denervated musculature. The distal stump of the transected tibial nerve is identified, trimmed and the peroneal nerve is mobilized at its distal end and microsurgically repaired to the tibial nerve stump. The peroneal nerve will grow at a rate of approximately 1 mm/day into the tibial nerve stump to reinnervate the gastrocnemius and soleus muscles. The tibial nerve transection model provides an advantage over the sciatic nerve transection model in that a nerve graft is not necessary in the reinnervation procedure2 because of the availability of the peroneal nerve. However, it should be noted that nerve reanastomoses requires the precision of a skilled and experienced operator.
In summary, here we demonstrate the tibial nerve transection model in mice, as an easy, robust, well validated and reproducible model of denervation-induced skeletal muscle atrophy.
The authors have nothing to disclose.
This work was supported by grants from the CIHR Neuromuscular Research Partnership (JNM – 90959; to J.A.E.B).
Reagents and Materials | |||
10-0 Nylon suture | Ethicon | 2850G | |
5-0 Vicryl suture | Ethicon | J553G | |
Equipment | |||
Spring microdissecting scissors | Fine Surgical Tools | 15021-15 | |
Ultra fine forceps | Fine Surgical Tools | 11370-40 | |
Non locking micro needle holder (driver) | Fine Surgical Tools | 12076-12 | |
Spring retractor | Fine Surgical Tools | 17000-02 |