Synergistic protective effects of 3,4-dihydroxyphenylglycol and hydroxytyrosol in male rats against induced heat stress-induced reproduction damage.

Testicular heat stress disrupts spermiogenesis and damages testicular tissue. The study aims to assess 3,4-dihydroxyphenylglycol (DHPG) and hydroxytyrosol (HT) from olive oil as antioxidants to reduce heat-induced testicular damage. Seven groups of 35 male rats were used. Group I got normal saline. Group 2 had HS (43 °C for 20 min/day) and normal saline for 60 days. Groups 3-7 had HS and DHPG/HT doses (0.5 mg/kg DHPG, 1 mg/kg DHPG, 5 mg/kg HT, 0.5 mg/kg DHPG + 5 mg/kg HT, and 1 mg/kg DHPG + 5 mg/kg HT). The evaluation included tests on testicular tissue, sperm quality, oxidative status, gene activity, and fertility after 60 days. After DHPG and HT treatment, sperm motility, viability, and plasma membrane functionality, as well as levels of total antioxidant capacity (TAC), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), and Bcl-2 gene expression, and in vivo fertility indexes increased. Meanwhile, abnormal morphology and DNA damage decreased, along with levels of glutathione (GSH), nitric oxide (NO), and malondialdehyde (MDA), and Bax, caspase-3, and caspase-9 gene expression, compared to the HS group. The study found that DHPG and HT have a more substantial synergistic effect when used together, improving reproductive health.

Improved Healing of Colonic Anastomosis with Allotransplantation of Axillary Skin Fibroblasts in Rats.

Objective: Colonic anastomosis is associated with serious complications leading to significant morbidity and mortality. Fibroblasts have recently been introduced as a practical alternative to stem cells because of their differentiation capacity, anti-inflammatory, and regenerative properties. The aim of this study was to evaluate the effects of intramural injection of fibroblasts on the healing of colonic anastomosis in rats. Materials and Methods: Inbred mature male Wistar rats were used in this experimental study (n=36). Fibroblasts were isolated from the axillary skin of a donor rat. In the sham group, manipulation on descending colon was done during laparotomy. A 5 mm segment of the colon was resected, and end-to-end anastomosis was performed. In the control group, 0.5 ml of phosphate buffer saline (PBS) was injected into the colonic wall and in the treatment group, 1×106 fibroblasts were transplanted. Following euthanasia on day 7, intra-abdominal adhesion, leakage and peritonitis were evaluated by necropsy. Mechanical properties were assessed using bursting pressure and tensile tests. Inflammation, angiogenesis, and collagen deposition were examined histopathologically. Results: The mean scores for adhesion and leakage were decreased in the treatment group versus control samples. Lower infiltration of inflammatory cells was observed in the treatment group (P=0.03). Angiogenesis and collagen deposition scores were significantly increased in the fibroblast transplanted group (P=0.03). Tensile mechanical properties of the colon were significantly increased in the treatment group compared to the control sample (P=0.01). There was no significant difference between the control and treatment groups in terms of bursting pressure (P=0.10). Positive weight changes were found in sham and treatment groups, but the control rats lost weight after 7 days. Conclusion: The results suggested that allotransplantation of dermal fibroblasts could improve the necroscopic, histopathological, and biomechanical indices of colonic anastomosis repair in rats.

Allotransplantation of ascorbic acid-treated fibroblasts improves healing of excisional cutaneous wound in diabetic rats.

The purpose of this study was to evaluate the effects of ascorbic acid (AA)-treated fibroblasts transplantation on excisional diabetic wound healing. An excisional wound was created between the shoulders of streptozotocin-induced diabetic rats. On day three, 1 ml of PBS, 1 × 106 intact homologous fibroblasts, and 1 × 106 fibroblasts treated with 50 µM AA were injected subcutaneously around the wound edges in control, treatment-1 and treatment-2 groups, respectively. In the sham group, the wound was left intact. Wound area was measured by planimetry. On day 15, samples were harvested for histopathological examination and hydroxyproline content. Wound area in treatment-1 and - 2 groups was significantly decreased compared to other groups, on days 11 and 15. The hydroxyproline content was significantly lower in the control group compared to the other groups. Histopathology revealed significant increases in the number of neovessels, macrophages, lymphocytes and fibroblasts in the treatment-2 group compared to the other groups. Trichrome staining showed the highest level of collagen deposition and orientation in the treatment-2 group. In conclusion, allotransplantation of 50 µM AA-treated fibroblasts could result in progressive healing and improved reparative indices of excisional dermal wound in diabetic rats.

Cross-link between mitochondrial-dependent apoptosis and cell cycle checkpoint proteins after experimental torsion and detorsion in rats.

The current study assessed the cross-link between mitochondria-related apoptosis and cell cycle machinery systems during ischemia and reperfusion in a rat model of testicular torsion and detorsion. The Wistar male rats were divided into control, 1 h, 2 h, 4 h and 8 h testicular torsion-induced, and 1 h, 2 h, 4 h and 8 h testicular detorsion-induced groups. The Johnson's score was analyzed. The mRNA and protein contents of Bcl-2, Bax, Caspase-3, Cyclin D1, Cdk4, P21 and P53 were investigated by sqRT-PCR and immunohistochemical staining, respectively. The apoptosis index was analyzed by TUNEL staining. The mRNA levels of bax, p53, p21 and cyclin D1 were increased, and the mRNA levels of bcl-2 and cdk4 were decreased in torsion and reperfusion-induced groups, time-dependently. The caspase-3 mRNA was increased in torsion-induced and diminished in detorsion-induced groups. A time-dependent reduction in Bcl-2+, Caspase-3+, Cyclin D1+, Cdk4+ and P53+ and increment in P21+ cells distribution per mm2 of tissue were revealed after torsion and detorsion. The apoptosis index was increased after torsion and decreased after detorsion. In conclusion, torsion-induced severe DNA damage stimulates the cyclin D1, p53 and p21 mRNA expression while more than 8 h is needed to reveal them as protein content in testicular tissue. About detorsion, decreased Cyclin D1 and Cdk4 proteins and the P53-induced transcriptional effect on p21 expression, stimulates the p21 bind to cdk4 and consequent failure in Cyclin D1/Cdk4 complex formation. This situation in association with apoptotic genes results in spermatogenesis failure.

Tenogenic effects of silymarin following experimental Achilles tendon transection in rats.

Tendon healing is prolonged due to the small number of cells, poor circulation, and low metabolism. The optimal tendon healing and its complete functional recovery have always been a challenge for researchers. Silymarin possesses anti-inflammatory, anti-oxidant, analgesic, and regenerative properties. The present study aimed to investigate the effects of silymarin on healing the Achilles tendon in rats. Twenty-four male Wistar rats were divided into two groups of control and treatment. After surgical preparation, a complete transverse incision was made in the middle part of the Achilles tendon, and then a modified Kessler suture was placed. The control group received 1.00 mL normal saline for five consecutive days, and the treatment group received 50.00 mg kg-1 of silymarin suspended in 1.00 mL normal saline for five days, orally. During the experimental period, Achilles functional index (AFI) was recorded. Six weeks after surgery, sampling was done. Histopathologically, a significant increase in the density of collagen fibers and reduction in neovascularization and inflammatory cells infiltration were observed in the treatment group. The biomechanical evaluation showed a significant increase in tensile strength of the tendon in the treatment group compared to the control group. The AFI results were concomitant with the results stated above, indicating an improvement in the AFI of rats in the treatment group. The present study results showed that oral administration of silymarin improved tissue healing indices, biomechanical properties, and functional index, leading to optimal healing of experimental Achilles tendon injury in the rat.

Effects of atorvastatin and resveratrol against the experimental endometriosis; evidence for glucose and monocarboxylate transporters, neoangiogenesis.

The current study was conducted to investigate the therapeutic effects of atorvastatin (ATV) and resveratrol (RVT) in sole and simultaneous forms of administration against the symbiosis between glucose transporters 1 and 3 (GLUT-1 and GLUT-3), monocarboxylate transporters 1 a and 4 (MCT-1 and MCT-4) and neovascularization in ectopic endometrial tissue (EET). For this purpose, the experimental endometriosis was induced in 24 virgin female Wistar rats, and then the rats were divided into non-treated endometriosis-induced (ENDO-sole), AVT-treated (5 mg kg-1), RVT-treated (40 mg kg-1) and AVT +RVT-treated groups (n = 6 rats in each group). Following 28 days from the experimental endometriosis induction, the EETs were collected and the EETs size, neovascularization ratio, and expression levels of GLUT-1, GLUT-3, MCT-1, and MCT-4 were analyzed by qRT-PCR and immunohistochemistry (IHC). The AVT and RVT sole and simultaneous-treated animals exhibited decreased EET sizes and neovascularization. Moreover, the mRNA levels of GLUT-1, GLUT-3, MCT-1, and MCT-4, as well as GLUT-1+, GLUT-3+, and MCT-4+ cells distribution per mm2 of tissue were decreased in AVT and RVT sole and simultaneous-treated groups. Our findings showed that the AVT and RVT, especially in the simultaneous form of administration, could decrease the neovascularization development in the EETs by suppressing the GLUTs (1 and 3) and MCTs (1 and 4) expressions. Therefore, it can be concluded that the simultaneous administration of AVT and RVT can inhibit the EET's establishment and development through suppressing glycolysis and neovascularization.

Evaluation of bilateral vasocystostomy for canine sterilization.

This study was aimed to evaluate canine vasocystotomy as a testosterone-preserving method of sterilization and investigate its potential post-operative complications. Five healthy adult male dogs underwent surgical procedure to transplant vasa deferentia to the urinary bladder. Under general anesthesia, caudal abdomen was opened and both vasa deferentia were ligated and transected. Then, the proximal free ends were sutured to mucosal layer of urinary bladder on its cranio-dorsal aspect. Serum testosterone level was measured on a weekly basis. Six-week postoperative assessments were performed including semen and urine sampling, ultrasound, contrast vasography, and tissue sampling. Statistical analyses revealed no significant differences in serum testosterone levels compared to its baseline value. Along with non-motile and broken spermatozoa, no cast or crystals were observed in urine samples. Semen analyses revealed azoospermia. No vasal obstruction or contrast leakage was observed in vasographs indicating bilateral patency in all dogs. Normal thickness of the bladder was found in ultrasounds. Histopathology showed normal testicular architecture and no inflammatory response was found in bladder or vas deferens following vasal transplantation. No significant change was found in testicular volume at the end of the study. This study suggested that vasocystostomy could be considered as an alternative method for canine sterilization with no significant changes in the testosterone concentrations and no evidence of postoperative complications. The preservation of testosterone could be regarded as an advantage and makes this approach favorable compared to the routine methods of sterilization especially for herding and guard dogs, because it prevents overpopulation while maintains the functionality.

Effects of rabbit pinna-derived blastema cells on tendon healing.

OBJECTIVES: Tendon healing is substantially slow and often associated with suboptimal repair. Cell therapy is one of the promising methods to improve tendon repair. Blastema, a population of undifferentiated cells, represents characteristics of pluripotent mesenchymal stem cells and has the potentials to be used in regenerative medicine. The aim of this study was to investigate the use of blastema allotransplantation in rabbit tendon healing. MATERIALS AND METHODS: In this study, one rabbit was used as a blastema donor, and twenty-four rabbits were divided into control and treatment groups. Blastema cells were obtained from ear pinna upon punch hole injury in the donor rabbit. Under general anesthesia, a complete transverse tenotomy was performed on the midsubstance of deep digital flexor tendon followed by suture-repair. In the treatment group, 1 × 106 blastema cells suspended in buffer saline were injected intratendinously at the repair site, while the control group received only the buffer saline. Cast coaptation was maintained for two weeks. Eight weeks after the operation, tendons were harvested, and histopathological, biomechanical, and biochemical assays were performed on samples. RESULTS: Mechanical testing showed a significant increase in ultimate load, energy absorption, stiffness, yield load, stress, and strain in blastema-treated tendons compared to controls. Also, higher hydroxyproline content and improved collagen alignment along with lower inflammatory cell infiltration and decreased angiogenesis were observed in blastema-treated tendons. CONCLUSION: Increased levels of hydroxyproline and improved histopathological and biomechanical parameters in the treatment group suggest that blastema cells could be considered an adjunct to tendon repair in rabbits.

Fabrication of novel tubular scaffold for tendon repair from chitosan in combination with zinc oxide nanoparticles.

Chitosan bears numerous properties, such as biocompatibility, biodegradability and non-toxicity making it suitable for use in different biomedical fields. Zinc (Zn) is required for fibroblasts proliferation and collagen synthesis as essential elements of wound healing. Its nanoparticles are well known for their capability to enhance wound healing by cell adhesion and migration improvement through growth factors-mediated mechanisms. Poor blood supply and unique histological characteristics of tendon make its regeneration always slow. Also, adhesion formation between tendon and its surrounding tissues is another problem for neotendon to return to its normal structure and functional activities. In this study, a novel tubular scaffold of zinc oxide (ZnO) nanoparticles loaded chitosan has been fabricated for tendon repair. Experimental complete tenotomy of deep digital flexor tendon in a rabbit model was done and scaffolds were placed in the transected area after two ends suturing. After four and eight weeks, adhesion formation around the tendons and tissue reaction to the scaffolds were evaluated macroscopically. Inflammation, angiogenesis and collagen fibers arrangement were also analyzed in histopathological evaluations. After eight weeks, the scaffolds were absorbed completely, adhesions around the tendon were decreased and there was no sign of significant tissue reaction and/or infection in histopathological analyses. The reduced adhesion formation, improved gliding function and better histopathological characteristics suggest this scaffold application as a potential therapy in treatment of tendon acute injuries.

Testicular torsion and reperfusion: evidences for biochemical and molecular alterations.

This study was done in order to determine the molecular and biochemical alterations following testicular torsion (TT) and torsion-reperfusion (TR). For this purpose, 54 male Wistar rats were divided into five groups as control group (n = 6) and experimental group subjected to 1, 2, 4, and 8 h unilateral left torsion induction (n = 12 in each group). After induction of TT, testicular samples were collected from each group (n = 6), and the other six rats of each group underwent the same period of reperfusion after TT and then were sampled. Histological changes, the mRNA and protein expression of heat shock protein-70 (Hsp70), and caspase-3 were examined using reverse transcriptase-PCR (RT-PCR) and immunohistochemistry, respectively. Testicular total antioxidant capacity (TAC), glutathione peroxidase (GSH-px), and malondialdehyde (MDA) levels were evaluated. The mRNA damage and DNA fragmentation were assessed. The TT and TR significantly reduced differentiation and spermiogenesis indices (p < 0.05). The TT- and TR-induced groups exhibited a severe reduction in Hsp70 expression as well as remarkable enhancement in caspase-3 expression. The TAC and GSH-px levels were decreased and the MDA content was increased in TT- and TR-induced groups. Finally, the TT and TR enhanced mRNA damage and DNA fragmentation. The TT- and TR-induced damaging oxidative stress, diminished Hsp70 expression, and enhanced caspase-3 mRNA and protein levels result in apoptosis following 1, 2, and 4 h. Whereas, following 8 h, TT and TR initiate the necrosis by inducing energy depletion as well as severe mRNA damage.

Effects of Unilateral Iatrogenic Vas Deferens Trauma on Fertility: An Experimental In Vitro Fertilization Mice Model Study.

OBJECTIVE: To determine bilateral effects of unilateral iatrogenic vas deferens trauma (UIT) on epididymal sperm characteristics and in vitro fertilizing capacity in an experimental mouse model. METHODS: Experiments were performed on three equal groups each comprising six adult male mice. Following anaesthesia, UIT was induced by clamping left vas deferens with a mosquito clamp in fully locked fashion for 2 minutes in UIT group. Control-sham mice only had exposure of the left vas deferens through a groin incision. Control animals only received ceftriaxone (100 mg/kg) intraperitoneally at the day of experimental UIT induction. Ipsilateral and contralateral epididymal sperm characteristics and in vitro fertilizing capacity were evaluated after 35 days. RESULTS: UIT significantly decreased sperm concentration, motility and viability as well as fertilization, two-cell embryos, blastocysts and hatched blastocysts rates. Moreover, incidence of DNA damage and abnormality in spermatozoa was significantly higher in UIT group. CONCLUSION: The findings suggest that a non-recognized iatrogenic vas deferens trauma may have detrimental effects on spermatozoa leading to infertility.

Comparative study on functional effects of allotransplantation of bone marrow stromal cells and adipose derived stromal vascular fraction on tendon repair: a biomechanical study in rabbits.

OBJECTIVE: Tendon never returns to its complete biological and mechanical properties after repair. Bone marrow and, recently, adipose tissue have been used as sources of mesenchymal stem cells which have been proven to enhance tendon healing. In the present study, we compared the effects of allotransplantation of bone marrow derived mesenchymal stromal cells (BMSCs) and adipose derived stromal vascular fraction (SVF) on tendon mechanical properties after experimentally induced flexor tendon transection. MATERIALS AND METHODS: In this experimental study, we used 48 adult male New Zealand white rabbits. Twelve of rabbits were used as donors of bone marrow and adipose tissue, the rest were divided into control and treatment groups. The injury model was a unilateral complete transection of the deep digital flexor tendon. Immediately after suture repair, 4×10(6)cells of either fresh SVF from enzymatic digestion of adipose tissue or cultured BMSCs were intratendinously injected into tendon stumps in the treatment groups. Controls received phosphate-buffered saline (PBS). Immobilization with a cast was continued for two weeks after surgery. Animals were sacrificed three and eight weeks after surgery and tendons underwent mechanical evaluations. The differences among the groups were analyzed using the analysis of variance (ANOVA) test followed by Tukey's multiple comparisons test. RESULTS: Stromal cell transplantation resulted in a significant increase in ultimate and yield loads, energy absorption, and stress of repairs compared to the controls. However, there were no statistically significant changes detected in terms of stiffness. In comparison, we observed no significant differences at the third week between SVF and BMSCs treated tendons in terms of all load related properties. However, at the eighth week SVF transplantation resulted in significantly increased energy absorption, stress and stiffness compared to BMSCs. CONCLUSION: The enhanced biomechanical properties of repairs in this study advocates the application of adipose derived SVF as an excellent source of multipotent cells instead of traditional BMSCs and may seem more encouraging in cell-based therapy for tendon injuries.

Effects of flunixin meglumine on experimental tendon wound healing: A histopathological and mechanical study in rabbits.

Tendons are frequently targets of injury in sports and work. Whether nonsteroidal anti-inflammatory drugs (NSAIDs) have beneficial effects on tendon healing is still a matter of debate. This study was conducted to evaluate effects of flunixin meglumine (FM) on tendon healing after experimentally induced acute trauma. Twenty eight adult male New Zealand White rabbits were subjected to complete transection of deep digital flexor tendons followed by suture placement. Treatment group received intramuscular injection of FM for three days, and controls received placebo. Subsequently, cast immobilization was continued for two weeks. Animals were sacrificed four weeks after surgery and tissue samples were taken. The histological evaluations revealed improved structural characteristics of neotendon formation including fibrillar linearity, fibrillar continuity and neovascularization in treatment group compared to those of controls (p < 0.05). However, no significant differences were found between two groups in terms of epitenon thickness (p > 0.05). Mechanical evaluation revealed significant increase in load-related material properties including ultimate load, yield load, energy absorption and ultimate stress in treatment group compared to those of control group (p < 0.05). However, no statistically significant differences in terms of stiffness and ultimate strain were found (p > 0.05). The present study showed that intramuscular injection of FM resulted in improved structural and mechanical properties of tendon repairs and it could be an effective treatment for acute tendon injuries like severance and laceration.

Enhanced mechanical properties of rabbit flexor tendons in response to intratendinous injection of adipose derived stromal vascular fraction.

INTRODUCTION: Tendon injuries are notorious for slow and functionally inferior healing. It is claimed that cell-based therapy would result in faster and more efficient healing of injured tissues with less postoperative complications. Given the limitations associated with ex vivo cellular expansion, we tried to evaluate the possible effects of intratendinous injection of adipose derived stromal vascular fraction on mechanical properties of tendon repair. METHODS: The model of injury was complete sharp transection of rabbit deep digital flexor tendon followed by primary suture repair and an intratendinous injection of either allogeneic stromal vascular fraction or placebo. Tendons were harvested at three and eight weeks after surgery. RESULTS: The results of mechanical testing showed the treatment caused significant increase in ultimate and yield loads, stress, and energy absorption of repairs compared to controls at both time points. Also, improvement in terms of strain and stiffness were detected at the eighth week in treatments. DISCUSSION: In comparison with the result of previous studies using cultured mesenchymal stem cells from bone marrow or adipose tissue; the improved mechanical properties observed in the present study suggest that choosing stromal vascular fraction as a readily accessible and instant source of multipotent cells instead of expensive and long-lasting culture expansion may seem more favorable in cell based therapy for tendon injuries. The mechanical functionality of the repairs observed in the present study encourages further investigations into the use of stromal vascular fraction for the repair of tendon injuries.

Adipose-derived stromal vascular fraction improves tendon healing in rabbits.

OBJECTIVE: To evaluate the potential effects of uncultured adipose-derived stromal vascular fraction on tendon healing. METHODS: Twenty five adult male New Zealand white rabbits weighing 2.5-3.0 kg were used. Five rabbits were used as donors of adipose tissue and the rest were divided into control and treatment groups. The injury model was completed by unilateral tenotomy through the middle one third of deep digital flexor tendon. Immediately after suture repair, either fresh stromal vascular fraction from enzymatic digestion of adipose tissue or placebo was intratendinously injected at tendon stumps in treatment and control groups, respectively. Immobilization with cast was continued for two weeks after surgery. Animals were sacrificed at eight weeks after surgery and tendons underwent histological, immunohistochemical, and mechanical evaluations. Statistical analyses of quantitative and qualitative data were assessed using one-way analysis of variance and Mann-Whitney U-test, respectively. RESULTS: Histological evaluations demonstrated superior fibrillar linearity and continuity, and decreased vascularity in treatment group indicated improved organization and remodeling of neotendons. Immunohistochemistry de- monstrated a significant increase in collagen I expression in treatment group. Ultimate load and energy absorption capacity were both significantly increased in cell-treated repairs compared with controls. CONCLUSION: The present study shows that intratendinous injection of uncultured adipose-derived stromal vascular fraction results in improved structural and mechanical properties of tendon repairs and it could be an effective modality for treating tendon injury.