Douglas E. Dow

Number of contractions to maintain mass and force of a denervated rat muscle

Within 5 weeks, denervated extensor digitorum longus (EDL) muscles of rats lose 66% of mass, 91% of force, and 76% of fiber cross‐sectional area (CSA). We previously determined the parameters of electrical stimulation for denervated rat EDL muscles to generate tetanic contractions sufficient to maintain mass and force close to control values. Using these parameters, we tested the hypothesis that a range exists for number of contractions per day, below and above which values for mass, maximum force, and fiber CSA are lower than values for innervated control muscles. For 5 weeks, denervated EDL muscles were stimulated to generate between 25 and 5000 contractions daily with contractions separated by constant intervals of rest, repeated 24 h per day. Force was not maintained with fewer than 200 or more than 800 contractions daily, whereas mass and fiber CSA were not maintained with fewer than 50 contractions daily. Protocols of stimulation that maintain muscle mass and force during prolonged periods of denervation may minimize problems clinically associated with denervation atrophy. Muscle Nerve 30: 77–86, 2004

An implantable device for stimulation of denervated muscles in rats

The purposes of the present study were (1) to develop an implantable device capable of being pre-programmed to generate a protocol of chronic contractions in denervated hind-limb muscles of rats, and (2) to verify the design by implanting the stimulators for five weeks in rats to identify a protocol of stimulation that maintains muscle mass and maximum force in stimulated-denervated extensor digitorum longus (EDL) muscles. This implantable stimulator system did not hinder animal movement or hygiene, and enabled the animals to be housed in regular animal facilities, since neither external equipment nor an externally generated magnetic field was required. The pre-programmable microcontroller allows detailed basic research into the cellular and tissue response to different stimulation protocols. The micropower design of the battery powered device enabled chronic stimulation of denervated EDL muscles for the five weeks of this initial study. Stimulation protocols of 9-11 V pulse amplitude, 0.4 ms bipolar pulse width, 100 Hz, 20 pulses per contraction, and 100 or 300 contractions generated per day maintained muscle mass and maximum force in denervated EDL muscles of rats at values near control values for innervated muscles.

Tissue-engineered axially vascularized contractile skeletal muscle

Background: As tissue-engineered muscle constructs increase in scale, their size is limited by the need for a vascular supply. In this work, the authors demonstrate a method of producing three-dimensional contractile skeletal muscles in vivo by incorporating an axial vascular pedicle. Methods: Primary myoblast cultures were generated from adult F344 rat soleus muscle. The cells were suspended in a fibrinogen hydrogel contained within cylindrical silicone chambers, and situated around the femoral vessels in isogeneic adult recipient rats. The constructs were allowed to incubate in vivo for 3 weeks, at which point they were explanted and subjected to isometric force measurements and histologic evaluation. Results: The resulting three-dimensional engineered skeletal muscle constructs produced longitudinal contractile force when electrically stimulated. Length-tension, force-voltage, and force-frequency relationships were similar to those found in developing skeletal muscle. Desmin staining demonstrated that individual myoblasts had undergone fusion to form multinucleated myotubes. Von Willebrand staining showed that the local environment within the chamber was richly angiogenic, and capillaries had grown into and throughout the constructs from the femoral artery and vein. Conclusions: Three-dimensional, vascularized skeletal muscle can be engineered in vivo. The resulting tissues have histologic and functional properties consistent with native skeletal muscle.

Electrical stimulation protocol to maintain mass and contractile force in denervated muscles

Due to atrophy, rat EDL muscles denervated for 1 month have a mass of only 38% of contralateral control muscles, and at 2 months it is down to 32%. Likewise, contractile force at 1 month is only 6% and at 2 months it is down to 2% of control values. Chronic electrical stimulation maintains both the muscles mass and contractile force at some percent of control values depending on the protocol used. We tested a stimulation protocol resembling the bursts of activity followed by periods of rest observed in animal behavior. We tested 2 protocols producing either 100 or 300 muscle contractions per day. EDL muscles of Wistar rats were denervated and stimulated for 5 weeks. The muscles mass and force were significantly different between the two groups. The 300 contractions/day protocol maintained both muscle mass and force above 80% of control values, whereas with 100 contractions/day the values did not differ significantly from unstimulated muscles.

Skeletal muscle construct in vascularized cylindrical chamber

A construct containing functional skeletal muscle tissue was formed in vivo within an axially vascularized chamber seeded with myoblasts suspended in a gel. After a three week period of incubation within a rat, the construct was harvested and evaluated for force generation capability. Constructs that were seeded with myoblasts generated a positive component of force (to shorten the construct), having contractile properties similar to those of skeletal muscles.