Antiproliferative and Mitochondrial Protective Effects of Apigenin in an Oxygen-Induced Retinopathy In Vivo Mouse Model.

Purpose: To investigate the effects of a common dietary flavonoid apigenin on retinal endothelial cell proliferation, retinal morphological structure, and apoptotic cell death in an oxygen-induced retinopathy (OIR) mouse model to evaluate the possibility of the use of apigenin in the treatment of ocular neovascular diseases (ONDs). Methods: Ninety-six newborn C57BL/6J mice were included. Eight groups were randomized, each including 12 mice. Two negative control groups were kept in room air: the first without any injection and the second received intravitreal (IV) dimethyl sulfoxide (DMSO), which is the solvent we used. The OIR groups were exposed to 75% ± 2% oxygen from postnatal days (PD) 7 to 12. On PD 12, the mice were randomly assigned to 6 groups: 2 OIR control groups (1 received no injection, 1 received IV-DMSO), 2 IV-apigenin groups (10 and 20 µg/mL), and 2 intraperitoneal (IP)-apigenin groups (10 and 20 mg/kg). We quantified retinal endothelial cell proliferation by counting neovascular tufts in cross-sections and examined histological and ultrastructural changes through light and electron microscopy. We evaluated apoptosis by terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL). Results: We detected a significant increase in endothelial cell proliferation in the OIR groups. Groups receiving apigenin, both IP and IV, had significant decreases in endothelial cells, atypical mitochondrion count, and apoptotic cells compared with the groups receiving no injections. None of the apigenin-injected groups revealed cystic degeneration or cell loss. Conclusions: Apigenin suppresses neovascularization, has antiapoptotic and antioxidative effects in an OIR mouse model, and can be considered a promising agent for treating OND. Clinical trial (Project number: DA15/19).

Comparative effects of photobiomodulation therapy at wavelengths of 660 and 808 nm on regeneration of inferior alveolar nerve in rats following crush injury.

The aim of the present study was to investigate the therapeutic effects of 660-nm and 880-nm photobiomodulation therapy (PBMT) following inferior alveolar nerve (IAN) crush injury. Following the nerve crush injuries of IAN, 36 Wistar rats were randomly divided into three groups as follows: (1) control, (2) 660-nm PBMT, and (3) 808-nm PBMT (GaAlAs laser, 100 J/cm2, 70 mW, 0.028-cm2 beam). PBMT was started immediately after surgery and performed once every 3 days during the postoperative period. At the end of the 30-day treatment period, histopathological and histomorphometric evaluations of tissue sections were made under a light and electron microscope. The ratio of the inner axonal diameter to the total outer axonal diameter (g-ratio) and the number of axons per square micrometer were evaluated. In the 808-nm PBMT group, the number of nerve fibers with suboptimal g-ratio ranges of 0-0.49 (p < 0.001) is significantly lower than expected, which indicates better rate of myelinization in the 808-nm PBMT group. The number of axons per square micrometer was significantly higher in the 808-nm PBMT group when compared with the control (p < 0.001) and 660-nm PBMT group (p = 0.010). The data and the histopathological investigations suggest that the PBMT with the 808-nm wavelength along with its settings was able to enhance IAN regeneration after nerve crush injury.

Evaluation of the Effects of Low-Level Laser Therapy on Diabetic Bone Healing.

The aim of the present study was to evaluate the effects of low-level laser therapy (LLLT) and biphasic alloplastic bone graft material on diabetic bone healing. Induction of diabetes was performed in 14 male Sprague-Dawley rats by intraperitoneal injection of a 50 mg/kg dose of streptozotocin. Two bilaterally symmetrical non-critical-sized bone defects were created in the parietal bones in each rat. Right defects were filled with biphasic alloplastic bone graft. Rats were randomly divided into 2 groups, with 1 group receiving 10 sessions of LLLT (GaAlAs, 78.5 J/cm, 100mW, 0.028 cm beam). The LLLT was started immediately after surgery and once every 3 days during postoperative period. At the end of treatment period, new bone formation and osteoblast density were determined using histomorphometry. Empty (control), graft-filled, LLLT-treated and both graft-filled and LLLT-treated bone defects were compared. New bone formation was higher in the graft treatment samples compared with the control (P = 0.009) and laser samples (P = 0.029). In addition, graft-laser combination treatment samples revealed higher bone formation than control (P = 0.008) and laser (P = 0.026) samples. Osteoblast density was significantly higher in the laser treatment (P <0.001), graft treatment (P = 0.001) and graft-laser combination treatment (P <0.001) samples than control samples. In addition, significantly higher osteoblast density was observed in the graft-laser combination treatment samples compared to the graft treatment samples (P = 0.005). The LLLT was effective to stimulate osteoblastogenesis but failed to increase bone formation. Graft augmentation for treatment of bone defects seems essential for proper bone healing in diabetes, regeneration may be supported by the LLLT to enhance osteoblastogenesis.

Effects of Platelet-Rich Fibrin Membrane on Sciatic Nerve Regeneration.

Alternative treatment approaches to improve the regeneration capacity of damaged peripheral nerves are currently under investigation. The objective of the present study was to evaluate the effects of platelet-rich fibrin (PRF) membrane after sciatic nerve crush injury in rabbits by histomorphometric and electromyographic analysis. The left sciatic nerves of 20 male Vienna rabbits were clamped for 30 seconds to induce crush injuries. Animals were randomly divided into 2 groups: PRF and control. For each animal in the PRF group, a PRF membrane was wrapped around the injured part of the sciatic nerve to form a tube. No additional treatment was performed in the control group. After a 12-week healing period, tissue samples from the injured nerve region were harvested and the g-ratio of axons, axon density, and impulse transmission changes were evaluated. Analysis revealed that axon density differences were not statistically significant between groups (P = 0.139). The rate of nerve fibers with optimum g-ratio was significantly lower in the PRF group than in the control group (P = 0.02). Conduction velocity differences between groups were not statistically significant. Although PRF application has previously shown positive regeneration effects on maxillofacial tissues, local PRF membrane application in tube form did not show any histomorphometric or functional improvement in peripheral nerve crush injury recovery.

Comparative evaluation of the electrophysiological, functional and ultrastructural effects of alpha lipoic acid and cyanocobalamin administration in a rat model of sciatic nerve injury.

BACKGROUND: Vitamin B12 and alpha lipoic acid (ALA) are known to promote functional and morphological recovery after peripheral nerve injury. OBJECTIVE: To compare the regenerative and neuroprotective effects of vitamin B12 and ALA treatment after sciatic nerve injury. METHODS: A total of 40 rats were randomly assigned to control (sciatic nerve exposure without injury or anastomosis), sham (sciatic nerve injury and epineural anastomosis were performed but no treatment was administered), PS (isotonic saline was administered for 12 weeks after surgery), ALA (2 mg/kg ALA was administered for 12 weeks after surgery), and vitamin B12 groups (2 mg/kg cyanocobalamin was administered for 12 weeks after surgery). Functional recovery was determined by footprint analysis, in vivo neurophysiology, and ex vivo histopathological examination. RESULTS: ALA treatment produced significant improvements in sciatic functional index values and non-significant improvements on electroneuromyography compared to vitamin B12 treatment. Upon histopathological examination, the regenerative effects of ALA were relevant to axonal structural recovery whereas vitamin B12 produced greater improvements in edema and myelination. CONCLUSIONS: While both vitamin B12 and ALA produced improvements after sciatic nerve injury, ALA was more functionally effective. The unique ultrastructural effects of vitamin B12 and ALA treatment should be considered in future studies.

Adipose-Derived Stem Cells Enhance Axonal Regeneration through Cross-Facial Nerve Grafting in a Rat Model of Facial Paralysis.

BACKGROUND: Cross-face nerve grafting combined with functional muscle transplantation has become the standard in reconstructing an emotionally controlled smile in complete irreversible facial palsy. However, the efficacy of this procedure depends on the ability of regenerating axons to breach two nerve coaptations and reinnervate endplates in denervated muscle. The current study tested the hypothesis that adipose-derived stem cells would enhance axonal regeneration through a cross-facial nerve graft and thereby enhance recovery of the facial nerve function. METHODS: Twelve rats underwent transection of the right facial nerve, and cross-facial nerve grafting using the sciatic nerve as an interpositional graft, with coaptations to the ipsilateral and contralateral buccal branches, was carried out. Rats were divided equally into two groups: a grafted but nontreated control group and a grafted and adipose-derived stem cell-treated group. Three months after surgery, biometric and electrophysiologic assessments of vibrissae movements were performed. Histologically, the spectra of fiber density, myelin sheath thickness, fiber diameter, and g ratio of the nerve were analyzed. Immunohistochemical staining was performed for the evaluation of acetylcholine in the neuromuscular junctions. RESULTS: The data from the biometric and electrophysiologic analysis of vibrissae movements, immunohistochemical analysis, and histologic assessment of the nerve showed that adipose-derived stem cells significantly enhanced axonal regeneration through the graft. CONCLUSION: These observations suggest that adipose-derived stem cells could be a clinically translatable route toward new methods to enhance recovery after cross-facial nerve grafting.

Biochemical and histopathological effects of catechin on experimental peripheral nerve injuries.

AIM: Catechin is a type of polyphenol, along with epicatechin, epigallocatechin, and epigallocatechin-gallate (EGCG). This study aims to investigate the effect of EGCG, a major metabolite of catechin, which is the principle bioactive compound in green tea, on rats with peripheral nerve injury. MATERIAL AND METHODS: A total of 74 rats were divided into six groups, namely the control, the trauma, the normal saline, a 25mg/kg EGCG, a 50mg/kg EGCG and a daily consumption group (10mg/kg EGCG was given intraperitoneally for 14 days before the trauma). Except the first group, the other groups underwent a 1-minute sciatic nerve compression by clip with 50gr/cm2 pressure. Nerve samples were obtained at 28 day after trauma for the biochemical and histopathological analysis. RESULTS: Our study showed that the Daily consumption, 25mg/kg EGCG and 50mg/kg EGCG groups demonstrated statistically significant decreased lipid peroxidation levels and particularly daily consumption, and the 25mg/kg EGCG group showed a favourable reduction of degeneration and edema histologically. CONCLUSION: This study shows that Catechin and its derivatives have a protective effect on peripheral nerve injury.

Bio-engineered synovial membrane to prevent tendon adhesions in rabbit flexor tendon model.

During tendon injuries, the tendon sheath is also damaged. This study aims to test effectiveness of engineered tendon synovial cell biomembrane on prevention of adhesions. Forty New Zealand Rabbits enrolled into four study groups. Engineered synovial sheath was produced by culturing cell suspension on fabricated collagen matrix membrane. Study groups were: tendon repair (group A), tendon repair zone covered with plane matrix (Group B), synovial suspension injection into the zone of repair over matrix (Group C), and biomembrane application (Group D). Biomechanical evaluations of tendon excursion, metacarpophalangeal and proximal interphalangeal joints range of motion, H&E and Alcian Blue with neutral red staining, and adhesion formation graded for histological assessments were studied. Ten non-operated extremities used as control. Tendon excursions and range of motions were significantly higher and close to control group for Group D, p < 0.05. Adhesion formation was not different among Groups C, D, and Control, p > 0.005. Hyaluronic acid synthesis was demonstrated at groups C and D at the zone of injury. Application of synovial cells into the tendon repair zone either by cell suspension or within a biomembrane significantly decreases the adhesion formation. Barrier effect of collagen matrix and restoration of hyaluronic acid synthesis can explain the possible mechanism of action.

Prostaglandin F receptor expression in intrauterine tissues of pregnant rats.

In this investigation, we studied the expression and localization of rat prostaglandin F (FP) receptor in uterine tissues of rats on gestational Days 10, 15, 18, 20, 21, 21.5 and postpartal Days 1 and 3 using Western blotting analysis, real-time PCR, and immunohistochemistry. A high level of immunoreactivity was observed on gestational Days 20, 21, and 21.5 with the most significant signals found on Day 20. FP receptor protein was expressed starting on gestational Day 15, and a fluctuating unsteady increase was observed until delivery. Uterine FP receptor mRNA levels were low between Days 10 and 18 of gestation (p < 0.05). The transcript level increased significantly on Day 20 and peaked on Day 21.5 just before labor (p < 0.05). There was a positive correlation between FP receptor mRNA expression and serum estradiol levels (rs = 0.78; p < 0.01) along with serum estradiol/progesterone ratios (rs = 0.79; p < 0.01). In summary, we observed an increase FP receptor expression in rat uterus with advancing gestation, a marked elevation of expression at term, and a concominant decrease during the postpartum period. These findings indicate a role for uterine FP receptors in the mediation of uterine contractility at term.

Subclinical left ventricular systolic dysfunction in patients with mild-to-moderate rheumatic mitral stenosis and normal left ventricular ejection fraction: an observational study.

OBJECTIVE: Mitral valve stenosis (MS) remains as an important cause of morbidity despite evolution in diagnosis and treatment. Generally, left ventricular (LV) systolic functions are well preserved in patients with MS. However, there are some studies showing impaired LV systolic functions in patients with pure MS. The purpose of this study was to evaluate subclinical LV systolic dysfunction in a cohort of isolated mild-to-moderate MS patients with normal LV ejection fraction (EF) by using tissue Doppler imaging (TDI) and velocity vector imaging (VVI) techniques. METHODS: Fifty patients with isolated mild-to-moderate MS (84% female, mean age 49.1±10.0 years) and 60 healthy subjects (76.7% female, mean age 49.1±10.5) were included in this cross-sectional observational study. Conventional echocardiography, TDI, strain (S) and strain rate (SRs) analysis were performed in all patients. RESULTS: Transmitral mean pressure gradient was 6.4±3.0 mmHg and mean mitral valve area was 1.45±0.36 cm² in patients with MS. Both longitudinal and circumferential S and SRs were significantly reduced in patients with MS (p<0.001). TDI-derived parameters myocardial acceleration during isovolumic contraction (IVA) and peak velocity during systolic ejection (Sa) were also significantly decreased in patients with isolated MS (p<0.001). LV ejection fraction (EF) was not correlated with deformation indices. Deformation parameters were not correlated with transmitral gradient or mitral valve area. CONCLUSION: VVI-derived deformation parameters may identify subclinical systolic dysfunction in patients with isolated MS with normal EF. These findings may give way to optimal timing for mitral valve surgery.

Tobacco-induced neuronal degeneration via cotinine in rats subjected to experimental spinal cord injury.

OBJECTIVES: Cigarette smoke contains over 4000 chemicals including well-characterized toxicants and carcinogens, among which is cotinine. Cotinine is the principal metabolite of nicotine that has adverse affects on the microcirculation via vasoconstriction, hypoxia and the wound-healing cascade. Its impact on spinal cord injury (SCI) has not been investigated yet. The aim of the present study is to investigate the cotinine effect on SCI. METHODS: 48 male Wistar rats were divided into six groups as follows: sham-control, sham-trauma, vehicle-control, vehicle-trauma, cotinine-control, and cotinine-trauma. Initially, a defined concentration of cotinine blood level was maintained by daily intraperitoneal injection of cotinine for 14 days in the cotinine groups. The concentration was similar to the cotinine dose in the blood level of heavy smokers. Only ethyl alcohol was injected in the vehicle groups during the same period. Then, SCI was performed by a Tator clip. The cotinine groups were compared with rats subjected to vehicle and sham groups by immunohistochemical biomarkers such as glial fibrillary acidic protein (GFAP) and 2,3-cyclic nucleotide 3-phosphodiesterase (CNP) expressions. Electron microscopic examination was also performed. RESULTS: GFAP-positive cells were noted to be localized around degenerated astrocytes. Marked vacuolization with perivascular and perineural edema was seen in the cotinin consumption groups. These findings showed the inhibition of regeneration after SCI. Similarly, vacuolization within myelin layers was noted in the cotinine groups, which was detected through reduced CNP expression. CONCLUSION: Cotinine, a main metabolite of nicotine, has harmful effects on SCI via GFAP and CNP expression. The findings of the present study support the hypothesis that tobacco causes neuronal degeneration via cotinine.

Mesenchymal stem cell therapy in necrotizing enterocolitis: a rat study.

We evaluated the potential therapeutic use of exogenous human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in an experimental rat model of necrotizing enterocolitis (NEC). Thirty-six newborn Sprague-Dawley rats were randomly divided into three groups: NEC, NEC + hBM-MSC, and a control (control and control + hBM-MSC). NEC was induced by enteral formula feeding, exposure to hypoxia-hyperoxia, and cold stress. After NEC was induced, iron-labeled hBM-MSCs were administered by intraperitoneal injection. All pups were killed on the fourth day following injection, and the terminal ileum was excised for a histopathological and immunohistochemical evaluation. The pups in the NEC + hBM-MSC group showed significant weight gains and improvements in their clinical sickness scores (p < 0.01). Bowel damage severity observed in the histopathological evaluation was significantly lower in the NEC + hBM-MSC group than that in the NEC group (p = 0.012). The number of MSCs homing to the bowel was significantly higher in the NEC + hBM-MSC group than that in the control + hBM-MSC group. In conclusion, this is the first study that has evaluated the effectiveness of hBM-MSCs in a neonatal rat NEC model. MSCs reduced histopathological damage significantly.

The morphological evaluation of ipsilateral and contralateral vasa deferentia in a rat model of unilateral spermatic cord torsion.

AIM: Spermatic cord torsion is a surgical emergency that requires early intervention to protect the effected testicle. The literature review about this ischemic reperfusion (I/R) injury reveals not only ipsilateral, but also contralateral testicular and epididymal injuries in a broad fashion. However, there is no data about vas deferens injury related with this surgical emergency. The aim of the study is to evaluate the morphological changes of the vas deferens due to testicular I/R injury. MATERIALS AND METHODS: Eighteen Wistar-Albino rats were allocated to three groups. Bilateral vasa deferentia of control group (Gr C, n = 6) were harvested without any surgical intervention. The torsion group was subjected to 2 h torsion and 2 h detorsion of the left testicle (Gr T, n = 6) and the third group underwent sham operations (Gr S, n = 6). Bilateral vasa deferentia of Gr T and S were harvested after surgery. The either side of the vas deferens was divided into three equal segments and these regions (adjacent to urinary bladder, medial and adjacent to testicle) were evaluated histopathologically. RESULTS: The electron microscopic evaluation of bilateral vasa deferentia of Gr T revealed different degrees of degeneration on either side. The region adjacent to testicle of the contralateral vas deferens was the most effected segment when compared with the other segments. CONCLUSION: In the light of these findings, it can be said that testicular I/R injury effects not only testis and epididymis, but also the adjacent vas deferens. This effect seems to be bilateral, like the testis and epididymis injury. Moreover, it mostly seems to depend on the apoptotic processes.

High dose cotinine may induce neural tube defects in a chick embryo model.

AIM: Nicotine is a well-known agent among 4000 chemicals in cigarettes. About 70 to 80% of nicotine is converted to cotinine, a major metabolite. The aim of the present study is to investigate the effect of cotinine on neural tube development in a chick embryo model. MATERIAL AND METHODS: Sixty fertile, specific pathogen free eggs were divided into 6 groups for this study. In the first group, a fixed cotinine concentration for each egg was calculated just to simulate the concentration of a smoker's blood level. A second experimental group was designed at a higher cotinine concentration. Embryos that succeeded to reach Hamburger-Hamilton stage 12 from each group were then embedded into paraffin for permanent sections. These two groups were compared with eggs subjected to vehicle (standard alcohol and ten times more alcohol concentration) and control groups (saline and sham groups). RESULTS: Embryos of the cotinine (regular dose), vehicle and control groups were normal, but embryos subjected to higher cotinine concentrations were malformed at the cranial part of the thoracic neural tube. CONCLUSION: Association of cotinine with neural tube defects was demonstrated in the present study. Cigarette smoking may induce hazardous effects on neural tube development.

Synovial cell culture and tissue engineering of a tendon synovial cell biomembrane.

Restrictive adhesions are a common complication of tendon injury and repair in the hand, resulting in severe dysfunction. Creating a barrier between the repair sites and surrounding tissue layers may prevent adhesions. We present the first stage in the process of developing a synovial biomembrane for this purpose. Synovial cells harvested from the Achilles tendon sheath and the knee joint of a Wistar albino rat were cultured for 2 weeks in culture medium, and then impregnated into a collagen type 1 matrix for another 2 weeks. Cells originating from both tendon and synovium demonstrated cell growth and layer formation on the surfaces of the matrix 2 weeks after impregnation. Alcian blue staining using Scott's method demonstrated the presence of acidic mucopolysaccharide, indicating hyaluronic acid (HA) production. This provides indirect evidence of functioning synovial cells on the membrane. It is possible to culture synovial cells and engineer a synoviocyte-collagen membrane that synthesizes endogenous HA. Application of this biomembrane to tendon repair sites may help to prevent adhesions after tendon repairs. Evaluation of this method on in vivo models is required.