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

In order to quantify the amount of new tissue formation a scoring method was used on standardized images of histological sections using an arbitrary scale of + 1 to + 4, where;

+4 >1,000 μM cartilage deposition and woven bone formation at the amputated bone end

The distribution of electrically stimulated, sham and control samples for each score value was analyzed graphically by % stacked column graph (X-cell, Microsoft office for Windows).

The number of vessels, in a standardized field of a histological section, stained with AB&OG, was counted using light microscopy and image analysis software (NIS-Elements 4.4, Nikon GmbH, Germany). Assessments were performed in blinded specimens examined in random order. Three 1 mm2 areas at the distal end of the stump where identified and vessels were counted by an independent observer (main measure/count option), blinded to the group setup. The mean number of vessels within the 3 areas was calculated and the means were subsequently used for statistical analysis.

Paraffin embedded, 7 μm thick sections of decalcified specimens were incubated with monoclonal mouse anti-human Neurofilament protein antibodies, which cross react with both human and rat neurofilament proteins (Clone 2F11, culture supernatant, 1:100; DAKO, Germany). An Isotype identical (IgG1) non-specific mouse antibody served as a negative control (eBioscience, Germany). For signal detection, an EnVision+ System-HRP (AEC) kit (Dako, Germany) was applied. Finally, a counterstain with hematoxylin was performed. Three slides per animal were analyzed using light microscopy (at 10x) and image analysis software.

Cell proliferation was measured with the thymidine analog BrdU (5-Bromo-2′-deoxyuridine) following its incorporation into newly synthesized DNA and its subsequent detection with an anti-BrdU antibody. Animals were administered sterile BrdU labeling reagent (intraperitoneal 1 ml/100 g body weight) (Life Technologies). Injections were performed on day 27 (1 day prior to harvesting the 28 day stumps) in 8 experimental animals, 8 controls, and 8 sham animals (Table 1) and at day 6 (1 day prior to harvesting the 7 day stumps) in 2 control and 2 experimental animals (Table 1). Stumps were collected, fixed, and decalcified as described above. Paraffin embedded 7 μm thick sections of the decalcified specimens were incubated with biotinylated monoclonal anti-BrdU antibodies according to the manufacture’s protocol (BrdU staining kit, Invitrogen). Quantitative evaluation of BrdU labeled cells was performed in a standardized image of histological sections using light microscopy (Ti-E, Nikon GmbH, Germany) and image analysis software (NIS-Elements 4.4, Nikon GmbH, Germany). Assessments were performed with blinded specimens examined in random order. Three 0,5 mm2 areas were chosen in the distal end of the stump. Labeled cells were marked by an independent observer (main measure/count option), blinded to the group setup, and the mean number of positive stained cells within the 3 areas was calculated. These means were subsequently used for statistical analysis.

Data were analyzed using Unifactorial analysis of variance ANOVA, (group number = 3), BiAS for WindowsTM version 11.0 software (http://www.bias-online.de). Cohen’s effect size d: d = 0,2 minimal effect; d = 0,5 middle effect; d > 0,8 bigger effect. Data are presented as mean ± SD and significance level was set at P < 0,05.

How to cite this article: Leppik, L. P. et al. Effects of electrical stimulation on rat limb regeneration, a new look at an old model. Sci. Rep. 5, 18353; doi: 10.1038/srep18353 (2015).