Effects of electrical stimulation on rat limb regeneration, a new look at an old model

Abstract

Limb loss is a devastating disability and while current treatments provide aesthetic and functional restoration, they are associated with complications and risks. The optimal solution would be to harness the body's regenerative capabilities to regrow new limbs. Several methods have been tried to regrow limbs in mammals, but none have succeeded. One such attempt, in the early 1970s, used electrical stimulation and demonstrated partial limb regeneration. Several researchers reproduced these findings, applying low voltage DC electrical stimulation to the stumps of amputated rat forelimbs reporting "blastema, and new bone, bone marrow, cartilage, nerve, skin, muscle and epiphyseal plate formation". In spite of these encouraging results this research was discontinued. Recently there has been renewed interest in studying electrical stimulation, primarily at a cellular and subcellular level, and studies have demonstrated changes in stem cell behavior with increased proliferation, differentiation, matrix formation and migration, all important in tissue regeneration. We applied electrical stimulation, in vivo, to the stumps of amputated rat limbs and observed significant new bone, cartilage and vessel formation and prevention of neuroma formation. These findings demonstrate that electricity stimulates tissue regeneration and form the basis for further research leading to possible new treatments for regenerating limbs.

Figure 1. Longitudinal histological sections of rat limb stumps.

All sections were stained with AB&OG. Approximate level of the amputation site is indicated with black dotted lines (a–f). Top row are electrically stimulated (a), sham (b) and control (c) stumps at 7 days after amputation (scale bar = 500 μM). Middle row are electrically stimulated (d), sham (e) and control (f) stumps at 28 days after amputation. Bottom row are high magnification (20x) (scale bar = 50 μM) of electrically stimulated (g), sham (h) and control (j) selected sites (black box) on stumps at 28 days after amputation. Significant cartilage formation is seen in the electrically stimulated (g) histological section versus scar formation in the sham (h) and control (j) sections.

Figure 2. Scores indicating osteocartilaginous formation in rat limb stumps.

Osteocartilaginous formation in electrically stimulated (white), sham (gray) and control (black) stumps at 7 (a) and 28 (b) days after amputation. Scores derived from a scale of +1 to +4, where +1 = no growth; +2 = growth <100 μM; +3 = growth between 100 and 1,000 μM, and +4 = growth >1,000 μM.

Figure 3. High magnification longitudinal histology section of electrically stimulated rat limb stump 28 days post amputation.

(a) Osteochondral ossification and vascularization (AB&OG staining, 20x, scale bar = 100 μM). Vascular ingrowth (black arrows). Reserve, proliferating cartilage, hypertrophy, calcification and newly formed bone areas are visible. (b) High magnification (40×; scale bar = 10 μM) of new vessels. (c) Graph indicating number of new vessels in 3 × 1 mm² square areas in rat limb stumps in all 3 groups 28 days post amputation. Significantly more new vessels are visible in electrically stimulated compared to sham (p < 0,05) and control (p < 0.01) stumps.

Figure 4. Neuroma formation in rat limb stumps at 7 and 28 days post amputation.

(Neurofilament protein staining, 20× magnification, scale bar = 50 μM). (a–c) No signs of neuroma formation in electrically stimulated (a), sham (b) and control (c) stumps 7 days post amputation. Neuroma formation (arrows) in sham (e) and control (f) stumps and no neuroma formation in electrically stimulated stump tissue (d) 28 days post amputation.

Figure 5. Drawing and photos of histological section of amputated rat limb stump, and ES device.

(a) Drawing of rat limb amputation with the amputation site (black dotted line) and implanted electrical stimulation device (D^ES). (b) Photo of the electrical stimulation device with a silver wire electrode loop (Ag) and platinum wire electrode (Pt) interposed with a 10 MΩ resistor (R). (c) Photo of longitudinal histological section of amputated limb stump, with the amputation site (black dotted line) and labeled bone marrow, muscle, humerus and scapula bones.