Experimental Study

Evaluation of the muscular alterations after the intra muscular application of ENDOPEEL

The authors studied the alteration of the gait and the tissue reaction of the muscle which got an endopeels injection.
We chose to apply endopeel into the pre tibial muscle of rats and study the histological alterations.
The choice of the pre tibial muscle of rats was made because of similarity as weight with the depressor muscle of the corner of the mouth in humans.

Objective : Study the biological phenomenon caused by the endopeels injection into the pre tibial muscle of the Wister Rat and evaluate the alterations in its gait and the histopathological alteration in the applied muscle.

Authors

Main Authors :
-Dr.Alain Tenenbaum
-Sr.Mauro Tiziani
Tomas Yung Joon Kim
Romulo Mené

01

Study
Group

Experimentators : R.Mené,M.Kim,A.Tenenbaum,M.Tiziani

N= 29 Wistar rats
Weight mean : 421g

Origin of animals : Biotério Central of Campus of Ribeirão Preto of São Paulo University
Maintenance : Experimental Pathology Lab of Pathology Department on Medicine School of Ribeirão Preto – USP
Animals keeping : polypropylen boxes
Animals food : basic diet of lab and water
Light conditions : natural light/dark cycles under controlled basic conditions
Experimentators : A.Tenenbaum,R.Mené,M.Kim,M.Tiziani

02

Animals Sacrifice & Histologic Samples

Conventional Microscopy

The mice were euthanized in CO2 chambers.

Incision of the skin with a blade nº 23
Finding and isolation of the pretibial muscle using Metzenbaum scissors
Section of tendons with a Mayo scissors.
Fixation of Portions of the muscles on sections of cork oak
Embedding using 50% formaldehyde solution - 10% buffered and 50% of pure paraffin for conventional optical microscopic study.
Staining Sections as routine histological process.
Microtome used : Leika RM 2155
Coloration of muscle slices with

-hematoxilin-eosin
-Masson´s trichrome
-and red picro-sirius.

Histologic examination was realized with a conventional microscope (Olympus BH2)
Observation under polarized lampe
Photographic documentation was realized with a Leika DMR microscope joined to a digital camera Leika CD 300F and compatible PC.

03

Material

6 rats/box

Animals were divided in 6 boxes, so that boxes from number 1 to 5 had 5 rats and box number 6 had 4 rats
All animals were submitted to a walking evaluation by footprint impression on special paper , and previous immersion of back legs on a soap solution.
Box 1 had the control group, that received the application of 0,1 ml saline solution ( NaCl 0,9%) on the right pretibial muscle.
The mice of boxes 2 to 5 received 0,1 ml of Endopeels Original Main Product on the same muscle as control group.
Box 6 was constituted of 4 rats, and each one received 0,5 ml of Endopeels original main product injected on the subcutaneous layer.
All the animals were submitted to walking evaluation before and after their respective injections.

04

Methods

Animals are sacrified after 10 days-1 month- 3 months-7 months

Gait Evaluation

To register the footprints of the rats, strips of paper measuring 43 cm long by 8.7 wide, sizes that are considered as being adequate for the rats ‘catwalk’ and built as per the De MEDINACELI; FREED; WYATT (1982) drawing. The paper was previously soaked in a solution of bromophenol blue were used.

For the preparation of the paper the bromophenol blue in its anhydrous (Sigma) form was diluted in at 1% in absolute acetone, acquiring an orange color.
The sheets of paper were soaked in this solution and placed in the sun to dry, then they were cut into strips and stored in plastic packages, so as to avoid humidity, which could alter the blue color.

The animals were made to walk on the catwalk covered with the previously prepared paper strips, with their hind legs soaked in a solution of neutral detergent, which immediately changes the color of the footprint from yellow to blue . The footprints remain on the paper strips, which were left to dry for the reading of the necessary measures to calculate the Sciatic Functional Index .
Later these were analyzed by a computer program (software) developed by SELLI (1998) , to obtain the Sciatic Functional Index that was proposed by BAIN; MACKINNON; HUNTER (1989), which takes into account the print length, the total toe spread between the 1st to the 4th toe, and the intermediate toes which are the 2nd and 3rd. The footprints of each animal were obtained before the application of endopeel, as a control, and immediately after the endopeel injection, and again, and at periodic intervals, when the animals were sacrificed for the extraction of the anterior tibia muscle for histological study.

The footprints were digitalized with a scanner and placed into the SELLI program, which allows for the capture, identification and analysis of the images, allowing for the application of other methods of identification of the Sciatic Functional Index (DE MEDINACELI, FREED & WYATT, 1982; DE MEDINACELI, DERENZO & WYATT, 1984; CARLTON & GOLDBERG, 1986), as well as Tibia Functionality indexes and the peroneal nerve (BAIN; MACKINNON & HUNTER, 1989). Once the footprint is shown on the monitor, its parameters are analyzed and measured, which require the placing of the cursor to be clicked in the correct predetermined sequence. The program then calculates and supplies the value of the index, and this is also stored in the computer.

Footprints of the mouse without any injection

Footprints of the mouse after 0.05 ml injection in the right pre tibial muscle

Sciatic Functional Index or SFI Calculation & Measurement

Schematic representation of the measurements realized to calculate the Sciatic Functional Index.

E: Experimental (operated foot);

N: Normal (opposite foot);

TS: Total opening of the toes (1st to 5th);

IT: Opening of the intermediate toes (2nd to 4th);

PL: Length of footstep

Program used to measure the Sciatic Functional Index SFI

Gait Analysis

Gait Conclusions

NaCl 0.9%, 0.1 ml into the pretibial muscle does not alter the gait.
Endopeel at 0.1 ml intramuscular pretibial affects the gait accentually, immediately after the application & moderately after 10 to 30 days.
The alteration in the gait caused by Endopeel decreases with time when applied via intramuscular, at this level of concentration.
Application into the subcutaneous tissue is not as efficient as intramuscular.
When 0.5 ml is applied into the subcutaneous tissue (pretibial muscle region) there is a progressive loss of gait, probably because of the slow diffusion of Endopeel from the subcutaneous tissue to the intra muscular one.