Photobiomodulation therapy was more effective than photobiomodulation plus arginine on accelerating wound healing in an animal model of delayed healing wound.

The combined and individual influences of photobiomodulation therapy (PBMT) and arginine on wound strength, stereological parameters, and gene expressions of some related growth factors in ischemic and delayed healing wounds in rats were analyzed. We divided 108 rats into six groups: control, lower energy density (LOW)-PBMT, 2% arginine ointment (Arg 2%), LOW-PBMT + Arg 2%, high energy density (HIGH)-PBMT, and HIGH-PBMT + Arg 2%. First, we generated an ischemic and delayed healing wound model in each rat. We examined wound strength, stereological parameters, and gene expressions of basic fibroblast growth factor (bFGF), vascular endothelial growth factor A (VEGF-A), and stromal cell-derived factor 1 (SDF-1) by quantitative real-time polymerase chain reaction (qRT-PCR). PBMT alone and PBMT + Arg 2% considerably increased wound strength compared to the control and Arg 2% groups during the inflammatory and proliferative steps of wound healing (p < 0.05). In these steps, PBMT alone significantly induced an anti-inflammatory effect and increased fibroblast counts; Arg 2% alone induced an inflammatory response (p < 0.05). Concurrently, PBMT and PBMT + Arg 2% significantly increased keratinocyte counts and volume of the new dermis (p < 0.05). At the remodeling step, the Arg 2% groups had significantly better wound strength than the other groups (p < 0.05). In this step, PBMT and PBMT + Arg 2% significantly decreased inflammation, and increased fibroblast counts, vascular length, and the volume of new epidermis and dermis compared to the control and Arg 2% groups (p < 0.05). In all cases of gene analysis, there were statistically better results in the PBMT and PBMT + Arg 2% groups compared with the Arg 2% and control groups (p < 0.05). The anti-inflammatory and repairing effects of PBMT on an ischemic and delayed healing wound model in rats were shown by significant improvements in wound strength, stereological parameters, and gene expressions of bFGF, VEGF-A, and SDF-1α.

Bone Marrow Mesenchymal Stem Cell Condition Medium Loaded on PCL Nanofibrous Scaffold Promoted Nerve Regeneration After Sciatic Nerve Transection in Male Rats.

Nowadays, researchers pay a vast deal of attention to neural tissue regeneration due to its tremendous effect on the patient's life. There are many strategies, from using conventional autologous nerve grafts to the newly developed methods for reconstructing damaged nerves. Among the various therapeutic methods, incorporating highly potent biomolecules and growth factors, the damaged nerve site would promote nerve regeneration. The aim was to examine the efficiency of a mesenchymal stem cell condition medium (MSC-CM) loaded on a 3D-polycaprolactone (PCL) scaffold as a nerve conduit in an axotomy rat model. Twenty-four mature male rats were classified into four groups: controls (the animals of this group were intact), axotomy (10 mm piece of the nerve was removed), axotomy (10-mm piece of the nerve was removed) + scaffold, and axotomy (10-mm piece of the nerve was removed) + MSC-CM-loaded scaffold. We followed up nerve motor function using a sciatic function index and electromyography activity of the gastrocnemius muscle. At 12 weeks post axotomy, sciatic nerve and dorsal root ganglion specimens and L4 and L5 spinal cord segments were separated from the rats and were analyzed by stereological, immunohistochemistry, and RT-PCR procedures. The rats of the axotomy group presented the expected gross locomotor deficit. Stereological parameters, immunohistochemistry of GFAP, and gene expression of S100, NGF, and BDNF were significantly enhanced in the CM-loaded scaffold group compared with the axotomy group. The most observed similarity was noted between the results of the control group and the CM-loaded scaffold group. Our results support the potential applicability of MSC-CM-loaded PCL nanofibrous scaffold to treat peripheral nerve injury (PNI).

The modified method of luxol fast blue for paraffin-embedded myelin sheath staining / El método modificado de luxol fast blue para la tinción de mielina fijada en parafina

SUMMARY: Histological techniques are the study of animal and human tissues through staining and examining them under a microscope. To demonstrate the axonal degeneration and demyelination in histological studies, the Luxol Fast Blue staining is gold standard techniques. In this study, a new histochemical method based on modified Luxol Fast Blue for the staining of the myelin sheath in sciatic nerve tissues described. The sciatic nerves of rats were removed and then the sciatic nerve was immersed in 10 % formaldehyde for one week and embedded in paraffin block. Next, thin sections (5 µm) were cut, using a microtome and stained with conventional and modified Luxol Fast Blue. Our results showed that a new method of modified Luxol Fast Blue staining can accurately identify the myelin in the sciatic nerve fibers. The current study showed that the Luxol Fast Blue combination with Light Green has a good effect on myelin coloration, and the results of this study are comparable to LFB combination with Sirius red.

RESUMEN: Las técnicas histológicas son el estudio de tejidos animales y humanos mediante tinción y examen bajo un microscopio. Para demostrar la degeneración axonal y la desmielinización en estudios histológicos, la tinción Luxol Fast Blue es una técnica estándar de oro. En este estudio, se describe un nuevo método histoquímico basado en Luxol Fast Blue modificado para la tinción de mielina en los tejidos del nervio ciático. Se seccionaron los nervios ciáticos de ratas y luego el nervio ciático se sumergió en formaldehído al 10 % durante una semana y se fijó en bloque de parafina. Posteriormente, se cortaron secciones delgadas (5 µm) usando un microtomo y se tiñeron con Luxol Fast Blue convencional y modificado. Nuestros resultados mostraron que un nuevo método de tinción Luxol Fast Blue modificado puede identificar con precisión la mielina en las fibras del nervio ciático. El estudio actual mostró que la combinación Luxol Fast Blue con Light Green es un buen efecto sobre la coloración de mielina, y los resultados de este estudio son comparables a la combinación LFB con Sirius red.

Sertoli cell-conditioned medium restores spermatogenesis in azoospermic mouse testis.

The current study evaluates potential applications of Sertoli cell (SC)-conditioned medium (CM) and explores the effects of the conditioned medium on the spermatogenesis process in azoospermic mice. For this study, 40 adult mice (28-30 g) were divided into 4 experimental groups: (1) control, (2) DMSO 2% (10 µl), (3) busulfan (40 mg/kg single dose) and (4) busulfan/CM (10 µl). SCs were isolated from 4-week-old mouse testes. After using anesthetics, 10 µl of CM was injected over 3-5 min into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. The data showed that CM notably increased the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocytes, round spermatids, SCs and Leydig cells compared with the control, DMSO and busulfan groups. Furthermore, the results showed that expression of c-kit and STRA8 was significantly decreased in the busulfan and busulfan/SC groups at 8 weeks after the last injection (p < 0.001) but no significant difference was found for PCNA compared with the control and DMSO groups (p < 0.05). These findings suggest that the Sertoli cell-conditioned medium may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine.

Ulmus minor bark hydro-alcoholic extract ameliorates histological parameters and testosterone level in an experimental model of PCOS rats.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is a common and multifactorial disease associated with female factor infertility. Ulmus minor bark (UMB) is one of the medicinal plants used in Persian folklore as a fertility enhancer. In the current study, we aimed to elucidate the effect of UMB hydro-alcoholic extract on histological parameters and testosterone condition in an experimental model of PCOS rats. METHODS: Thirty female rats were randomly divided into five groups: (1) control, (2) vehicle, (3) PCOS/50 mg [6 mg/kg dehydroepiandrosterone (DHEA) + 50 mg/kg UMB hydro-alcoholic extract], (4) PCOS/150 mg (6 mg/kg DHEA + 150 mg/kg UMB hydro-alcoholic extract), and (5) PCOS (6 mg/kg DHEA). All interventions were performed for 21 days. Afterwards, stereological analysis was done for determination of ovarian volume and follicle number. The serum level of testosterone was measured by ELISA kit. RESULTS: UMB hydro-alcoholic extract improved the total number of the corpus luteum in the treatment groups when compared to the PCOS group (p<0.05). PCOS/150 mg and PCOS/50 mg groups showed significantly lower total number of the primordial, primary, and secondary follicles as well as testosterone level compared to the PCOS group (p<0.05). The total number of antral follicles and volume of ovary did not differ significantly between groups. CONCLUSION: UMB extract may be an effective and good alternative in improving PCOS histo-logical and testosterone disturbances although further studies are warranted to confirm the safety of UMB plant in human.

Sciatic nerve injury alters the spatial arrangement of neurons and glial cells in the anterior horn of the spinal cord.

The spatial arrangement of the cell is important and considered as underlying mechanism for mathematical modeling of cell to cell interaction. The ability of cells to take on the characteristics of other cells in an organism, it is important to understand the dynamical behavior of the cells. This method implements experimental parameters of the cell-cell interaction into the mathematical simulation of cell arrangement. The purpose of this research was to explore the three-dimensional spatial distribution of anterior horn cells in the rat spinal cord to examine differences after sciatic nerve injury. Sixteen Sprague-Dawley male rats were assigned to control and axotomy groups. Twelve weeks after surgery, the anterior horn was removed for first- and second-order stereological studies. Second-order stereological techniques were applied to estimate the pair correlation and cross-correlation functions using a dipole probe superimposed onto the spinal cord sections. The findings revealed 7% and 36% reductions in the mean volume and total number of motoneurons, respectively, and a 25% increase in the neuroglial cell number in the axotomized rats compared to the control rats. In contrast, the anterior horn volume remained unchanged. The results also indicated a broader gap in the pair correlation curve for the motoneurons and neuroglial cells in the axotomized rats compared to the control rats. This finding shows a negative correlation for the distribution of motoneurons and neuroglial cells in the axotomized rats. The cross-correlation curve shows a negative correlation between the motoneurons and neuroglial cells in the axotomized rats. These findings suggest that cellular structural and functional changes after sciatic nerve injury lead to the alterations in the spatial arrangement of motoneurons and neuroglial cells, finally affecting the normal function of the central nervous system. The experimental protocol was reviewed and approved by the Animal Ethics Committee of Shahid Beheshti University of Medical Sciences (approval No. IR.SBMU.MSP.REC1395.375) on October 17, 2016.

Effects of Sertoli Cell Transplantation on Spermatogenesis in Azoospermic Mice.

BACKGROUND/AIMS: The aim of this study was to evaluate the potential and significant applications of Sertoli cells (SCs) transplantation, and to explore the effect of transplantation on spermatogenesis process, in azospermic mice. METHODS: In this study, we utilized 18 adult mice (28‒30 g), divided into four experimental groups: (1) control, (2) vehicle (DMSO 2%) (10 µl) (3) busulfan and (4) busulfan+ SCs (1×104 cells/µL). SCs were isolated from the testis of 4-week-old mouse and after using anesthetics, 10 µl of SCs suspension (1×104 cells/µL) was injected over 3-5 min, into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments, and RNA extraction, in order to examine the expression of c-kit, STRA8 and PCNA genes. RESULTS: Our data showed that SCs transplantation could notably increase the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocyte, round spermatid, SCs and Leydig cells, compared to the control, DMSO and busulfan groups. Furthermore, the result showed that the expression of c-kit and STRA8 were significantly decreased in busulfan and busulfan/SCs groups, at 8 weeks after the last injection (p<0.001), but no significant decrease was found for PCNA, compared to the control and DMSO groups (P<0.05). CONCLUSION: These findings suggest that SCs transplantation may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine. We further highlighted the essential applications that might provide a mechanism for correcting fertility in males, suffering from cell deformity.