Orhan Özatik

Effects of ischemic preconditioning protocols on skeletal muscle ischemia-reperfusion injury.

BACKGROUND Ischemic preconditioning (IPC) is described as brief ischemia-reperfusion (I/R) cycles to induce tolerance to subsequent in response to longer I/R insults. Various IPC protocols can be performed in four combinations as follows: at early or late phases and on local or distant organs. Although many experimental studies have been performed on IPC, no consensus has been established on which IPC protocol is most effective. The aims of the present study were as follows: (1) to compare the variables of preconditioning in different combinations (in early versus late phases; local versus remote organ implementations) and (2) to determine the most therapeutic IPC protocol(s). MATERIALS AND METHODS A subtotal hind limb amputation model with clamping an intact femoral pedicle was used for I/R injury. IPC was induced using hind limb tourniquet with 3 × 10 min I/R cycles before longer I/R insult. Forty-nine rats were divided into seven groups (n = 7), sham, IsO (ischemia only), I/R, early ischemic preconditioning (e-IPC), late ischemic preconditioning (l-IPC), early remote ischemic preconditioning (e-RIPC), and l-RIPC (late-remote) groups, respectively. In the sham group, pedicle occlusion was not performed. Six hours ischemia was challenged in the IsO group. Three hours ischemia followed by 3 h reperfusion was performed in the I/R group. The e-IPC group was immediately preconditioned, whereas the l-IPC group was preconditioned 24 h before I/R injury on the same hind limb. In the e-RIPC and l-RIPC groups, the same protocols were performed on the contralateral hind limb. At the end of the experiments, skeletal muscle tissue samples were obtained for biochemical analysis (Malondialdehyde [MDA], catalase, myeloperoxidase [MPO], and nitric oxide end products [NOx]), light microscopy, and caspase-3 immunohistochemistry for determination of apoptosis. RESULTS Tissue biochemical markers were improved in nearly all the IPC groups compared with IsO and I/R groups (P < 0.05). Similarly, the histologic damage scores were decreased in all the IPC groups (P < 0.05). The lowest damage score was in the e-RIPC group followed by the l-RIPC, e-IPC, and l-IPC groups, respectively. The apoptosis scores were significantly high in the I/R group compared with the e-RIPC and l-RIPC groups (P < 0.05). Although apoptosis scores of the e-IPC and l-IPC groups were lower than the I/R group, this finding was not statistically significant (P > 0.05). CONCLUSIONS All IPC protocols were effective in reducing I/R injury. Among these protocols, e-RIPC achieved most protection.

Effects of tadalafil on hemorrhagic cystitis and testicular dysfunction induced by cyclophosphamide in rats.

Introduction: The protective and/or therapeutic potential of tadalafil (TDL) on cyclophosphamide (CP)-induced hemorrhagic cystitis (HC) and testicular dysfunction in rats was evaluated. Materials and Methods: The animals except from the control group were divided into four groups and treated with saline, or 1, 5 or 10 mg/kg TDL orally (CP, TDL1, TDL5 and TDL10 groups, respectively) before and after CP injection. Body and organ weights, sperm count, cGMP, nitric oxide (NO), IL-6 and IL-10 levels in serum and bladder tissue, and serum testosterone (T), LH and FSH levels were determined. The histological analysis of bladder and testis was performed and the number of apoptotic cells was determined. Results and Conclusion: The CP group had decreased cGMP and NO levels in the bladder, serum T level (p < 0.05) and sperm count (p < 0.001) and higher IL-6 levels in serum and bladder (p < 0.01). Treatment with TDL resulted in increased cGMP (p < 0.001), NO (p < 0.05) and serum T (p < 0.05) levels. Histological analysis of the CP group showed severe HC in bladder and testicular damage. TDL-treated animals showed a dose-dependent improvement in all of these histological impairments. In conclusion, a selective inhibitor of phosphodiesterase-5 enzyme, TDL, showed a protective and/or therapeutic effect on CP-induced HC and testicular dysfunction in rats.

Comparison of the effects of curcumin, tramadol and surgical treatments on neuropathic pain induced by chronic constriction injury in rat

AIM Nerve entrapment syndromes are the most common causes of neuropathic pain. Surgical decompression is the preferred method of treatment. The aim of this study was to compare the efficacy of curcumin, tramadol and chronic constriction release treatment (CCR), individually or together, in a rat model of sciatic nerve injury. MATERIAL AND METHODS Eighty male rats were divided into eight study groups. Group 1 was the sham group. Group 2 was the control group with established chronic constriction injury (CCI). CCI was also established in Groups 3?8. Group 3 underwent chronic constriction release (CCR). Groups 4 and 5 received curcumin and tramadol. Groups 6 and 7 also received curcumin (100 mg/kg daily, oral) and tramadol (10 mg/kg daily, intraperitoneal, 14 days) after CCR, respectively. Combined curcumin-tramadol treatment was applied to Group 8. Behavioral tests (thermal hyperalgesia, dynamic plantar, cold plate test) were performed on days 0,3,7,13,17, and 21. Tissue tumor necrosis factor-? (TNF-?) and interleukin-10 (IL-10) levels were analyzed in the nerve and dorsal root ganglion (DRG) samples on day 21. Histopathological examination was performed on the nervous tissue and DRG. RESULTS Tramadol-CCR and tramadol-curcumin significantly attenuated mechanical allodynia and thermal hyperalgesia. In the CCI-CCR-tramadol treatment group, TNF-? levels were significantly lower in the sciatic nerve tissue, and DRG and IL-10 levels were significantly higher in the sciatic nerve tissue. CONCLUSION CCI-CCR-tramadol treatment is highly effective in the symptomatic treatment of neuropathic pain. CCR-curcumin is associated with less degeneration and high levels of regeneration in the nerve tissue.

The effects of iloprost and alprostadil on ischemia-reperfusion injury in preventing inflammation, tissue degeneration, and apoptosis in rat skeletal muscle

BACKGROUND/AIM The protective effects of prostaglandin (PG) analogs on ischemia-reperfusion (I/R) have been well documented; however, comparative studies are lacking. The aim of the present study was to determine whether iloprost or alprostadil is more effective in preventing muscle I/R injury. MATERIALS AND METHODS Thirty-two rats were divided into four groups (n = 8): sham, control, IL (I/R + iloprost), and AL (I/R + alprostadil). I/R was induced by a tourniquet in the hindlimb for 3 h/3 h. The IL and AL groups received iloprost (0.5 ng kg-1 min-1) and alprostadil (0.05 µg kg-1 min-1) during reperfusion, respectively. After 6 h, blood and muscles were collected for analyses. RESULTS Serum TNF-α and IL-1β levels were decreased in the IL and AL groups compared with the control group (P < 0.05), whereas IL-6 levels did not change significantly. Tissue malondialdehyde levels were significantly lower in the IL and AL groups (P < 0.05). Tissue catalase levels showed no difference. The histological damage scores and apoptosis scores were both significantly decreased in the IL and AL groups compared with the control group (P< 0.05). CONCLUSION The present study indicated that iloprost and alprostadil attenuated I/R injury in skeletal muscle. However, no comparable difference was evident regarding the efficacies of either PG analog.

Enhancement of vascular endothelial growth factor’s angiogenic capacity by the therapeutic modulation of notch signalling improves tram flap survival in rats submitted to nicotine

Abstract Background: Smoke of cigarettes, and specifically nicotine, has been shown to diminish pedicled transverse rectus abdominis musculocutaneous (TRAM) flap survival. Considering that Notch signalling through its ligand Delta-like 4 (Dll4) functions as anti-angiogenic factor by inhibiting the pro-angiogenic effects of vascular endothelial growth factor (VEGF), it is hypothesised that inhibition of the Notch would promote angiogenesis and increase TRAM flap survival in rats submitted to nicotine. Methods: Twenty rats were treated with nicotine for 28 days preoperatively. Thereafter, a pedicled TRAM flap was created in all animals. The Notch inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine-t-butyl-ester was administered in animals of the treatment group. Animals in the control group were given the same amount of solvent. Five days after the surgery, viable flap areas were determined. Skin samples were evaluated for VEGF and Dll4 mRNA levels. Immunohistochemical analysis was used for the assessment of endothelial Dll4 expression. Vascular density was determined histologically. Plasma levels of VEGF and Dll4 were measured. Results: A significant improvement in TRAM flap surviving area was observed in the treatment group (53.50 ± 14.25%) compared with the controls (32.20 ± 9.15%). Immunohistochemical analysis revealed a significant increase in the number of Dll4 stained vessels in animals of the treatment group (9.2 ± 1.6) in comparison with the controls (5.7 ± 1.9). VEGF mRNA levels (0.22 ± 0.08) in the treatment group were significantly lower than those in the control group (0.36 ± 0.09). Conclusion: Notch inhibition significantly improved TRAM flap survival in animals exposed to nicotine by promoting VEGF-induced angiogenesis.

Can a Small Intestine Segment Be an Alternative Biological Conduit for Peripheral Nerve Regeneration

Background: Autologous nerve grafts are used to bridge peripheral nerve defects. Limited sources and donor site morbidity are the major problems with peripheral nerve grafts. Although various types of autologous grafts such as arteries, veins and muscles have been recommended, an ideal conduit has not yet been described. Aims: To investigate the effectiveness of a small intestinal conduit for peripheral nerve defects. Study Design: Animal experimentation. Methods: Twenty-one rats were divided into three groups (n=7). Following anaesthesia, sciatic nerve exploration was performed in the Sham group. The 10 mm nerve gap was bridged with a 15 mm ileal segment in the small intestinal conduit group and the defect was replaced with orthotopic nerve in autologous nerve graft group. The functional recovery was tested monthly by walking-track analysis and the sciatic functional index. Histological evaluation was performed on the 12th week. Results: Sciatic functional index tests are better in autologous nerve graft group (-55.09±6.35); however, during follow-up, progress in sciatic functional index was demonstrated, along with axonal regeneration and innervation of target muscles in the small intestinal conduit group (-76.36±12.08) (p<0.05). In histologic sections, distinctive sciatic nerve regeneration was examined in the small intestinal conduit group. The expression of S-100 and neurofilament was observed in small intestinal conduit group but was less organised than in the autologous nerve graft group. Although the counted number (7459.79±1833.50 vs. 4226.51±1063.06 mm2), measured diameter [2.19 (2.15-2.88) vs. 1.74 (1.50-2.09) µm] and myelin sheath thickness [1.18 (1.09-1.44) vs. 0.66 (0.40-1.07) µm] of axons is significantly high in the middle sections of autologous nerve graft compared to the small intestinal conduit group, respectively (p<0.05), the peripheral nerve regeneration was also observed in the small intestinal conduit group. Conclusion: Small intestinal conduit should not be considered as an alternative to autologous nerve grafts in its current form; however, the results are promising. Even though the results are no better than autologous nerve grafts, with additional procedures, it might be a good alternative due to harvesting abundant sources without donor site morbidity.

NERVE TİSSUE PREFABRİCATİON İNSİDE THE RAT FEMORAL BONE: DOES İT WORK?

AIM To investigate whether nerve regeneration can be induced in the tubular bone between distal and proximal cut nerve ends. MATERIAL AND METHODS Twenty adult Wistar rats were used for the study. Rats were divided into three groups; femoral bone conduit group, nerve transection group, sham group. The sciatic nerve was surgically cut and from both ends inserted into the adjacent femoral bone tunnel in the femoral bone conduit group. The sciatic nerve was cut transversely in the nerve transection group. In the Sham group, only sciatic nerve exploration was performed, without a nerve cut. The groups were evaluated functionally and morphologically. RESULTS All results showed that axonal growth existed through the osseous canal. CONCLUSION To the best of our knowledge, this is the first study to evaluate neural regeneration inside the bone. We can speculate that the bone marrow provides a convenient microenvironment for peripheral nerve regeneration. In addition to prefabricating peripheral nerves, this novel model may help to establish further strategies for engineering of other tissues in the bone marrow.