# Assessment of Nerve Repair Augmented with Adipose-Derived Mast Cells in an Animal Model
## 2 Materials and Methods  
We used 20 Wistar male rats, weighing between 400 and 600 g, aged between 6 and 8 months. Sixteen animals formed the study group. Four rats (8 sciatic nerves) were used as reference for the normal sciatic nerve and gastrocnemius muscle--an indirect measure of nerve regeneration [10].  
The study animals were operated on using continuous general anesthesia (Isoflurane, [PERSON], Bucharest, Romania) by the same surgeon. A complete transection of the sciatic nerve was performed on both hind limbs followed by:  
* Direct neurorrahphy (end-to-end nerve repair) alone on the right side;
* Neurorrahphy plus processed fat autograft around the nerve on the left. The 16 rats (treatment group) were analyzed at 2 time points:
* The first 8 animals (S1-S8) were sacrificed at 4 weeks after nerve repair;
* The following 8 at 8 weeks.  
Animals were culled by injecting T61 (embutramide, Intervet/Schering-Plough Animal Health, NJ, USA). Nerve and gastrocnemius muscle biopsies were collected from both hind limbs (Figure 1). The authors used a similar model in a previous study [11].  
Approximately 3 g of fat tissue was harvested from the inguinal area and centrifuged for 3 min at 3000 rpm (1008\(\times\)_g_). This led to separation in three layers, from top to bottom:  
Figure 1: The control group and the two treatment arms.  
oil, processed fat, and the fluid portion. We used the processed fat to cover the nerve repair site on all left limbs.  
The tissue specimens were fixed in 10% buffered formalin for 24 h, followed by routine paraffin embedding. Three micrometer serial sections were performed, and one slide for each case was stained with hematoxylin and eosin for histopathological diagnosis. One slide from each case was stained by luxol fast blue histochemical method, following the manufacturer's instructions (Bio-Optica, Milano, Italy).  
Additional slides were selected to quantify CD34-positive (Abbiotec, San Diego, CA, USA) blood vessel cells as a measure of angiogenesis, together with mast cell trypatase (MCT) (Leica Microsystems, Wetzlar, Germany). All immunohistochemical procedures were performed automatically using the Bond Max System (Leica Microsystems, Wetzlar, Germany).  
On day 0, after anesthesia, we performed ultrasonography sizing of both (right and left) gastrocnemius muscles for each animal. The process was repeated at 4 weeks and 10 weeks. We recorded the maximum diameters (at their thickest part) using a Mindray M5 portable ultrasound with a linear piece 7L4S (Mindray, Shenzhen, China). Measurements were repeated 3 times during each session and a mean value of the diameter was used for each group, as previously described [10].  
Conventional microscopy was performed for the interpretation of morphology and immunohistochemistry stained slides by an AxioZoom Image A2 Research Microscope (Zeiss, Jena, Germany). Images were acquired by an AxioCam 506 Color and analyzed with ZEN software (Zeiss, Jena, Germany). Quantification method: select 3 consecutive microscopic fields with the highest number of mast cells and vascular density, count the number of blood vessels and mast cells at 20\(\times\) magnification, and determine the mean value. Descriptive statistics are presented using Microsoft Excel (Microsoft, Washington, DC, USA).