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Application of a low-level laser therapy and the purified protein from natural latex (Hevea brasiliensis) in the controlled crush injury of the sciatic nerve of rats: a morphological, quantitative, and ultrastructural study.

Dias FJ, Issa JP, Iyomasa MM, Coutinho-Netto J, Calzzani RA, Iyomasa DM, Sousa LG, de Almeida SR, Cury DP, Watanabe IS - Biomed Res Int (2013)

Bottom Line: After 4 weeks, the myelin density and morphological characteristics improved in groups LG, PG, and LPG compared to IG.After 8 weeks, PG, and LPG were similar to CG and the capillary density was higher in the LG, PG, and LPG.In the ultrastructural analysis the PG and LPG had characteristics that were similar to the CG.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil.

ABSTRACT
This study analyzed the effects of a low-level laser therapy (LLLT, 15 J/cm(2), 780 nm wavelength) and the natural latex protein (P1, 0.1%) in sciatic nerve after crush injury (15 Kgf, axonotmesis) in rats. Sixty rats (male, 250 g) were allocated into the 6 groups (n = 10): CG-control group; EG-nerve exposed; IG-injured nerve without treatment; LG-crushed nerve treated with LLLT; PG-injured nerve treated with P1; and LPG-injured nerve treated with LLLT and P1. After 4 or 8 weeks, the nerve samples were processed for morphological, histological quantification and ultrastructural analysis. After 4 weeks, the myelin density and morphological characteristics improved in groups LG, PG, and LPG compared to IG. After 8 weeks, PG, and LPG were similar to CG and the capillary density was higher in the LG, PG, and LPG. In the ultrastructural analysis the PG and LPG had characteristics that were similar to the CG. The application of LLLT and/or P1 improved the recovery from the nerve crush injury, and in the long term, the P1 protein was the better treatment used, since only the application of LLLT has not reached the same results, and these treatments applied together did not potentiate the recovery.

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Myelin density quantification: (a) original sciatic nerve photomicrograph; (b) photomicrograph of the sciatic nerve with the peripheral and blood capillaries areas cleared; (c) photomicrograph in binary (black and white) used during quantification.
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fig1: Myelin density quantification: (a) original sciatic nerve photomicrograph; (b) photomicrograph of the sciatic nerve with the peripheral and blood capillaries areas cleared; (c) photomicrograph in binary (black and white) used during quantification.

Mentions: The quantitative histological analysis of the myelin consisted of quantifying the number of pixels. In the original images (Figure 1(a)), the external and the capillaries areas have been erased (Figure 1(b)) using the software Photoshop CS, and this procedure was necessary since this dark staining in particular areas of the erythrocytes could be interpreted as myelin. The next step is to convert the cleaned images into binary form (black and white) (Figure 1(c)), which was performed using the ImageJ 1.45 s software, with the following commands “process/binary/make it binary,” or if you prefer to use Photoshop CS software commands “image/adjust/threshold,” in this case it is necessary to choose a threshold point (1–255), which once chosen, this value should be respected in all images analyzed; in this study the images were transformed into binary by Photoshop CS method with the threshold point of 128. The binary images in ImageJ software with the following command “plugins/analyze/area calculator” (area calculator plugin, free download at http://rsb.info.nih.gov/ij/plugins/area.html/, not included in original plugin pack of ImageJ) provide the area of the dark region in pixels2 representing myelin; if the program was calibrated to the scale of the microscope used in the capture of the images, it is possible to obtain the same area in μm2. For the realization of area ratio of myelin (myelin area/nerve area) it was also necessary to measure the cross-sectional area of the sciatic nerve, which was obtained from the original images (Figure 1(a)) using the command “Polygon selections” encircling the region of the nerve and then the command “analyze/measure.” The ratio was obtained by dividing the first value obtained in relation to the cross-sectional area of the nerve; these data were compared between groups. For the analysis of capillary density, blood vessels were counted and then the ratio of vessels/nerve area (capillaries/mm2) was compared among the groups. Statistical analysis of the quantitative analyses was performed using the SPSS 17.0 software, employing an analysis of variance (ANOVA, P < 0.05).


Application of a low-level laser therapy and the purified protein from natural latex (Hevea brasiliensis) in the controlled crush injury of the sciatic nerve of rats: a morphological, quantitative, and ultrastructural study.

Dias FJ, Issa JP, Iyomasa MM, Coutinho-Netto J, Calzzani RA, Iyomasa DM, Sousa LG, de Almeida SR, Cury DP, Watanabe IS - Biomed Res Int (2013)

Myelin density quantification: (a) original sciatic nerve photomicrograph; (b) photomicrograph of the sciatic nerve with the peripheral and blood capillaries areas cleared; (c) photomicrograph in binary (black and white) used during quantification.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3713596&req=5

fig1: Myelin density quantification: (a) original sciatic nerve photomicrograph; (b) photomicrograph of the sciatic nerve with the peripheral and blood capillaries areas cleared; (c) photomicrograph in binary (black and white) used during quantification.
Mentions: The quantitative histological analysis of the myelin consisted of quantifying the number of pixels. In the original images (Figure 1(a)), the external and the capillaries areas have been erased (Figure 1(b)) using the software Photoshop CS, and this procedure was necessary since this dark staining in particular areas of the erythrocytes could be interpreted as myelin. The next step is to convert the cleaned images into binary form (black and white) (Figure 1(c)), which was performed using the ImageJ 1.45 s software, with the following commands “process/binary/make it binary,” or if you prefer to use Photoshop CS software commands “image/adjust/threshold,” in this case it is necessary to choose a threshold point (1–255), which once chosen, this value should be respected in all images analyzed; in this study the images were transformed into binary by Photoshop CS method with the threshold point of 128. The binary images in ImageJ software with the following command “plugins/analyze/area calculator” (area calculator plugin, free download at http://rsb.info.nih.gov/ij/plugins/area.html/, not included in original plugin pack of ImageJ) provide the area of the dark region in pixels2 representing myelin; if the program was calibrated to the scale of the microscope used in the capture of the images, it is possible to obtain the same area in μm2. For the realization of area ratio of myelin (myelin area/nerve area) it was also necessary to measure the cross-sectional area of the sciatic nerve, which was obtained from the original images (Figure 1(a)) using the command “Polygon selections” encircling the region of the nerve and then the command “analyze/measure.” The ratio was obtained by dividing the first value obtained in relation to the cross-sectional area of the nerve; these data were compared between groups. For the analysis of capillary density, blood vessels were counted and then the ratio of vessels/nerve area (capillaries/mm2) was compared among the groups. Statistical analysis of the quantitative analyses was performed using the SPSS 17.0 software, employing an analysis of variance (ANOVA, P < 0.05).

Bottom Line: After 4 weeks, the myelin density and morphological characteristics improved in groups LG, PG, and LPG compared to IG.After 8 weeks, PG, and LPG were similar to CG and the capillary density was higher in the LG, PG, and LPG.In the ultrastructural analysis the PG and LPG had characteristics that were similar to the CG.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil.

ABSTRACT
This study analyzed the effects of a low-level laser therapy (LLLT, 15 J/cm(2), 780 nm wavelength) and the natural latex protein (P1, 0.1%) in sciatic nerve after crush injury (15 Kgf, axonotmesis) in rats. Sixty rats (male, 250 g) were allocated into the 6 groups (n = 10): CG-control group; EG-nerve exposed; IG-injured nerve without treatment; LG-crushed nerve treated with LLLT; PG-injured nerve treated with P1; and LPG-injured nerve treated with LLLT and P1. After 4 or 8 weeks, the nerve samples were processed for morphological, histological quantification and ultrastructural analysis. After 4 weeks, the myelin density and morphological characteristics improved in groups LG, PG, and LPG compared to IG. After 8 weeks, PG, and LPG were similar to CG and the capillary density was higher in the LG, PG, and LPG. In the ultrastructural analysis the PG and LPG had characteristics that were similar to the CG. The application of LLLT and/or P1 improved the recovery from the nerve crush injury, and in the long term, the P1 protein was the better treatment used, since only the application of LLLT has not reached the same results, and these treatments applied together did not potentiate the recovery.

Show MeSH
Related in: MedlinePlus