Evaluation and comparison of the effects of hyperbaric oxygen and ozonized oxygen as adjuvant treatments in an experimental osteomyelitis model.

BACKGROUND: We evaluated and compared the efficacy of ozone (O(3)) and hyperbaric oxygen (HBO) therapies in an experimental rat model of osteomyelitis. MATERIALS AND METHODS: Forty-eight male Sprague-Dawley rats were divided into sham, osteomyelitis (control), vancomycin (V), vancomycin + HBO (VHB), vancomycin + O(3) (VO), and vancomycin + HBO + O(3) (VOHB) groups. Osteomyelitis was induced by a bone injection of 10(8) CFU/mL methicillin-resistant Staphylococcus aureus. HBO was administered daily at 2.8-atm pressure for 90 min; O(3) therapy was provided as intraperitoneal injections of 0.7 mg/kg O(3)/O(2) gas mixture once daily. Treatments were continued from d 7 to 21 after induction of osteomyelitis. Bone tissues and blood samples were harvested for biochemical, histopathologic, and microbiologic analyses. RESULTS: Rats in the sham, VO, and VOHB groups gained weight but those in the control, V, and VHB groups did not. Levels of malondialdehyde, superoxide dismutase, and glutathione peroxidase were lower in the VHB, VO, and VOHB groups than in V and control groups. Levels of interleukin-10 and -1ß and tumor necrosis factor-α were decreased in the VHB, VO, and VOHB groups; transforming growth factor-ß was increased in these groups compared with V and control groups (P ≤ 0.001). Bacteria counts in VOHB were significantly lower than those in group of V (P = 0.012). Histopathologic scores in group VO were significantly lower than those in group V (P = 0.046). CONCLUSIONS: O(3) was as effective as HBO in decreasing oxidative parameters and inflammatory cytokines. Rats in the VO and VOHB groups gained more weight than did the other groups. Bacteria counts were significantly decreased in group VOHB compared with the other groups. Histopathologic scores in group VO were significantly decreased compared with the other groups.

Poly(ADP-ribose) polymerase inhibition modulates experimental acute necrotizing pancreatitis-induced oxidative stress, bacterial translocation and neopterin concentrations in rats.

Various studies have been performed to find out novel treatment strategies for acute necrotizing pancreatitis (ANP). Inhibition of poly(ADP-ribose) polymerase (PARP) is shown to reduce inflammation in several pathological conditions. We aimed to evaluate the efficacy of benzamide, a PARP inhibitor, in an experimental model of ANP. Thirty Sprague-Dawley rats were divided into three groups: sham-operated, ANP and ANP + benzamide groups. All groups except the sham-operated group were subjected to the ANP procedure, induced by infusing of 1 mL/kg of 3% sodium taurocholate into the common biliopancreatic duct. The ANP + benzamide group received 100 mg/kg/day benzamide intraperitoneally for a total of three days after induction of pancreatitis. The surviving animals were killed at the fourth day and the pancreas was harvested for biochemical, microbiological and histological analysis. Blood samples were also obtained from the animals. In the ANP group, a significant increase was observed in concentrations of serum amylase and neopterin and tissue oxidative stress indices (malondialdehyde, superoxide dismutase and glutathione peroxidase). Almost all of these changes were found to be reversed to near their normal values in the ANP + benzamide group. Histological injury scores were significantly higher in the ANP group than in the sham group (P < 0.05, ANP versus sham), and were significantly lower in the ANP + benzamide group than in the ANP group (P < 0.05, ANP + benzamide versus ANP). Evaluation of bacterial translocation identified significantly fewer infected sites in the ANP + benzamide group than in the ANP animals (P < 0.01). We observed that inhibition of PARP with benzamide reduced the severity, the mortality, the bacterial translocation rates and the neopterin concentrations in an experimental ANP model in rats. These findings suggest that it may be possible to improve the outcome of ANP by using PARP inhibitors.

Synergistic effects of hyperbaric oxygen and granulocytecolony stimulating factor on postoperative adhesion formation in a rat cecal abrasion model.

PURPOSE: We investigated the synergistic effect of hyperbaric oxygen (HBO) and granulocyte-colony stimulating factor (G-CSF) on adhesion formation in rats. METHODS: 40 adult male Sprague-Dawley rats (250-350 g) were divided into 4 groups. In group-1, no further management was undertaken. Group-2 received HBO therapy, group-3 was treated with 50 ug/kg subcutaneous G-CSF once daily for 7 days following laparatomy and cecal abrasion and group-4 was given both G-CSF and HBO therapies. On the 7th day, all rats were sacrificed and adhesions were scored. Tissue samples from adhesions and peritonea and cecum wall were examined both pathologically and biochemically for tissue hydroxyproline content. RESULTS: No mortality occurred in study groups. When the groups were evaluated according to the adhesion numbers and grades, there was a statistically significant difference between the control and groups 3 and 4 (P < 0.001). There was no statistically significant difference between groups 1 and 2 (p > 0.05). HBO + G-CSF group was significantly different from control, HBO and G-CSF groups, regarding hydroxyproline contents (p = 0.005). Inflammation and fibrosis did not differ significantly among the groups (p = 0.248), (p = 0.213). CONCLUSION: HBO treatment could not reduce the adhesion formation alone. Combined use of HBO and G-CSF, has a markedly preventive effect on postoperative adhesion formation (Tab. 1, Fig. 2, Ref. 57).

Melatonin prevents peritoneal adhesions in rats.

BACKGROUND AND AIM: Intra-abdominal adhesions are important postoperative complications following abdominal surgery. The adhesions that develop form the basis of more advanced pathology such as intestinal obstruction or infertility. Melatonin is secreted from the pineal gland in a circadian pattern; this molecule has potent antioxidant characteristics and has beneficial effects in many models of inflammation. The aim of this study was to evaluate the effects of melatonin on peritoneal adhesions created in rats. METHODS: A total of 28 Sprague-Dawley male rats were used and divided into four groups. In the first phase of the study, pinealectomy (PINX) was performed on half the animals. An incision was made and sutured in the cecum of all experimental animals in all groups 15 days after the PINX procedure. Some animals were given melatonin orally at a dose of 5 mg/kg daily following the adhesion operation and continued for 15 days. The rats were anesthetized and the abdomen opened after the 15th day (on day 30 of the study). After adhesion scoring based on macroscopic inspection, tissue samples were obtained from the sutured region of the cecum to measure malondialdehyde and hydroxyproline. RESULTS: Peritoneal adhesion density was significantly higher in the PINX group compared to the control animals; exogenously administered melatonin significantly reduced adhesion formation. The degree of adhesion was also significantly lower in the intact rats given melatonin compared to the control group. CONCLUSION: Antioxidant activity increases in the oxidative process. We conclude that melatonin may be an important molecule in preventing peritoneal adhesions.

The efficacy of ozone therapy in experimental caustic esophageal burn.

INTRODUCTION: Ozone has been proposed as an antioxidant enzyme activator, immunomodulator and cellular metabolic activator. This study was designed to investigate the efficacy of ozone therapy in the prevention of esophageal damage and stricture formation developed after esophageal caustic injuries in the rat. MATERIALS AND METHODS: Forty-five rats were allocated into three groups; sham-operated, un-treatment and treatment groups. Caustic esophageal burn was created by instilling 15% NaOH in the distal esophagus. The rats were left untreated or treated with 1 mg/kg/day ozone intraperitoneally. All rats were sacrificed at 28 days. Efficacy of the treatment was assessed by measuring the stenosis index (SI) and histopathologic damage score, and biochemically by determining tissue hydroxyproline content (HP), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) and protein carbonyl content (PCC) in esophageal homogenates. RESULTS: Whereas seven (47%) rats died in the un-treatment group, all rats in the sham-operated and the treatment group survived during the study. SI, the histopathologic damage score, was significantly lower in the ozone-therapy group than the un-treatment group. HP levels were significantly higher in the un-treatment group than the group treated with ozone. Caustic esophageal burn increased MDA and PCC levels and also decreased SOD and GPx enzyme activities. In contrast, ozone therapy decreased the elevated MDA and PCC levels and also increased the reduced SOD and GPx enzyme activities. CONCLUSION: Ozone has a preventive effect in the development of fibrosis by decreasing tissue damage and increasing the antioxidant enzyme activity in an experimental model of corrosive esophageal injury.

Retroperitoneal lymph node mapping with intratesticular injected patent blue dye in rats.

OBJECTIVES: Endolymphatic injection of several dyes have been previously studied to identify retroperitoneal lymphatic structure in animals and humans with malignant diseases. However, there have been no studies, to our knowledge, that demonstrate the utility of injecting patent blue dye into the testicular parenchyma to detect retroperitoneal lymphoid structure. The aim of this experimental study was to investigate whether intratesticular patent blue dye injection is feasible and is an accurate method for retroperitoneal lymph node mapping in rats. MATERIALS AND METHODS: Twenty male albino Wistar rats were included in the study and divided over two equal groups. The first group underwent patent blue violet (PBV) injection into the spermatic funiculus, while the second group underwent PBV injection into the testicular parenchyma. After the injection, the color changes in the retroperitoneal lymphatic structures and the urinary bladder were anticipated. The time interval between the injection and the staining of lymphatic structures and urinary bladder was measured for each intervention. Blue stained retroperitoneal nodal tissues were dissected and removed. These nodal tissues were examined histologically. RESULTS: After PBV injection, intense staining of the ipsilateral spermatic cord lymphatics was seen and anticipated color changes in the retroperitoneal lymphatic structures and urinary bladder were evaluated visually. Both application routes of dye resulted in the same distribution of retroperitoneal lymph nodes in the same time frame. All retroperitoneal nodular tissues removed were noted histologically to be lymph nodes and were found to be consistent with the ipsilateral lumbar lymph and the ipsilateral suprarenal lymph nodes according to the staining order in both groups. No toxic effects were observed histologically. There were no statistically significant differences in the time intervals between the two groups. CONCLUSIONS: We demonstrated that both funicular and intratesticular injections of patent blue dye are feasible and accurate methods for retroperitoneal lymph node mapping in rats. This shows that intralymphatic dye injection is not absolutely necessary to detect retroperitoneal lymphatic structures and may have applications beyond testis cancer.

Effects of poly(ADP-ribose) polymerase inhibition in bladder damage caused by cyclophosphamide in rats.

It was previously shown that nitric oxide produced by inducible nitric oxide synthase (iNOS) and peroxynitrite are responsible for cyclophosphamide (CP)-induced cystitis. Since endogenous production of peroxynitrite is known to lead to poly(ADP-ribose) polymerase (PARP) activation, in this study, the aim was to evaluate whether the PARP activation pathway is also included in the pathogenesis of CP-induced bladder ulceration in rats. A total of 48 male albino Wistar rats were divided into 5 groups. Group 1 served as control and was given 2 ml saline; four groups received a single dose of CP (200 mg/kg) with the same time intervals. Group 2 received CP only; Group 3, selective iNOS inhibitor 1400W (20 mg/kg); Group 4, peroxynitrite scavenger ebselen (30 mg/kg); and Group 5, PARP inhibitor 3-aminobenzamide (20 mg/kg). CP injection resulted in severe cystitis with continuous macroscopic hemorrhage, strong edema, inflammation, and ulceration. Moreover, bladder iNOS activation and urine nitrite-nitrate levels were dramatically increased. Histologically, 1400W protected bladder against CP damage and decreased urine nitrite-nitrate levels and bladder iNOS induction. Ebselen has shown similar histologic results with 1400W without changing urinary nitrite-nitrate level and iNOS activity. Furthermore in the 3-aminobenzamide group, beneficial effects had also occurred including decreased ulceration. These results suggest that PARP activation involves pathogenesis of CP-induced bladder ulceration. Furthermore, PARP is not only important for ulceration but also for bladder edema, hemorrhage, and inflammation because of broken uroepithelial cellular integrity.

Alpha-tocopherol, beta-carotene and melatonin administration protects cyclophosphamide-induced oxidative damage to bladder tissue in rats.

Cyclophosphamide (CP) has potential urotoxicity such as hemorrhagic cystitis (HC). 2-Mercaptoethane sulfonate (mesna) has been widely used as an effective agent against CP-induced cystitis, but significant HC has still been encountered clinically. In recent studies, mesna was shown to be more effective if combined with antioxidants. The purpose of this study was to evaluate the effects of antioxidants, alpha-tocopherol, beta-carotene and melatonin on CP-induced bladder damage in rats, even if used without mesna administration. Male Sprague-Dawley rats weighing 180-210 g were divided into 5 groups. Four groups received a single dose of CP (100 mg/kg) intraperitoneally with the same time intervals. Group 2 received CP only, group 3 received beta-carotene (40 mg/kg/day), group 4 received alpha-tocopherol (40 mg/kg/day) and group 5 received melatonin (10 mg/kg/day) both before and the day after CP injection. Group 1 served as control. Bladder histopathology, as well as malondialdehyde (MDA) and iNOS levels, and excretion of nitrite-nitrates (NO(x)) in urine were evaluated. CP injection resulted in severe histological changes and macroscopic hematuria. alpha-Tocopherol and melatonin showed meaningful protection against bladder damage. Protection by beta-carotene was also significant but weaker. MDA levels increased significantly with CP injection and all antioxidants ameliorated this increase in bladder tissue. CP also elevated the NO(x) level in urine and iNOS activity in bladder. Only melatonin was able to decrease these parameters. In conclusion, there is no doubt that oxidants have a role in the pathogenesis of CP-cystitis. Antioxidants, especially melatonin and alpha-tocopherol, may help to ameliorate bladder damage induced by CP.

Peroxynitrite may be involved in bladder damage caused by cyclophosphamide in rats.

PURPOSE: It was previously shown that nitric oxide (NO) produced by inducible NO synthase (iNOS) is responsible for cyclophosphamide (CP) induced cystitis. In this study we evaluated whether peroxynitrite is also responsible for CP induced bladder damage in rats. MATERIALS AND METHODS: A total of 38 male albino Wistar rats were divided into 4 groups. Group 1 served as controls and was given 2 ml saline, while 3 groups received a single dose of CP (200 mg/kg) at the same intervals. Group 2 received CP only, group 3 received the selective iNOS inhibitor aminoguanidine (AG) (100 mg/kg) and group 4 received the peroxynitrite scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) (20 mg/kg). RESULTS: CP injection resulted in severe cystitis with continuous macroscopic hemorrhage, strong edema, inflammation and ulceration. Moreover, bladder tissue malondialdehyde levels, iNOS activation and urine nitrite-nitrate levels were dramatically increased. AG histologically protected bladder against CP damage and decreased urine nitrite-nitrate levels, bladder malondialdehyde and iNOS induction. Ebselen showed results similar to those of AG without changing the urinary nitrite-nitrate level and iNOS activity. CONCLUSIONS: These results suggest that not only nitric oxide, but also peroxynitrite may be important in the pathogenesis of CP induced cystitis.

Melatonin ameliorates bladder damage induced by cyclophosphamide in rats.

Cyclophosphamide (CP), an alkylating antineoplastic agent, has potential urotoxicity including causing hemorrhagic cystitis (HC). HC is now accepted as a non-infectious inflammation and the pathogenesis of HC includes cytokine production which leads to inducible nitric oxide synthase (iNOS) induction. Moreover, overproduction of reactive oxygen species (ROS) during inflammation leads to extensive oxidative stress, cellular injury and apoptosis/necrosis via several mechanisms. Based on these facts, the aim of this study was to evaluate the protective effects of melatonin as an antioxidant, iNOS inhibitor and peroxynitrite scavenger against CP-induced urinary bladder damage. A total of 30 male Sprague-Dawley rats were divided into four groups. Three groups received a single dose of CP (100 mg/kg) intraperitoneally with the same times. Group 2 received CP only, group 3 received 5 mg/kg/day and group 4 received 10 mg/kg/day melatonin before and the day after CP administration. Group 1 served as the control. Increased iNOS induction, bladder malonyldialdehyde (MDA) levels and urinary nitrite-nitrate excretion were encountered in the CP-only group leading to severe cystitis. Melatonin exhibited significant protection against CP-induced cystitis by diminishing bladder oxidative stress and blocking iNOS and peroxynitrite production. Oxidants may have a major role in the pathogenesis of CP-induced cystitis and iNOS is an important mediator leading to peroxynitrite production. Melatonin ameliorates bladder damage induced by CP.

Exogenously administered and endogenously produced melatonin reduce hyperbaric oxygen-induced oxidative stress in rat lung.

Hyperbaric oxygen (HBO) is a widely used treatment modality in many diseases. A known side effect of HBO is the production of reactive oxygen species. Many antioxidants such as vitamins C and E, riboflavin and selenium have been used successfully to scavenge the reactive oxygen species caused by HBO administration. In this study, we aimed to see if melatonin, a newly discovered antioxidant, has a protective effect against the overproduction of reactive oxygen species produced by HBO in rat lung tissue. Sixty male Sprague-Dawley rats were divided into 5 groups as follows: control, daytime HBO (3 ATA, 120 min), daytime HBO plus melatonin (10 mg/kg), nighttime HBO and nighttime HBO (under light exposure). The MDA, SOD and CAT levels of daytime and nighttime HBO (under light exposure) increased significantly. This significance was not found in the daytime HBO plus melatonin and nighttime HBO groups when compared with the control. In this study, HBO caused oxidant stress, and melatonin decreased the levels of MDA, SOD and CAT. Moreover, endogenous melatonin was found to be a more effective antioxidant than exogenous 10 mg/kg melatonin.

Involvement of nitric oxide and hyperbaric oxygen in the pathogenesis of cyclophosphamide induced hemorrhagic cystitis in rats.

PURPOSE: We evaluated the relationship between nitric oxide and hyperbaric oxygenation in the pathogenesis and treatment of cyclophosphamide induced hemorrhagic cystitis in rats. MATERIALS AND METHODS: Cyclophosphamide (100 mg/kg) was injected in male Sprague-Dawley rats for cystitis induction. Animals were treated before and the day after cyclophosphamide injection with 100 mg/kg of the nitric oxide substrate L-arginine, 20 mg/kg of the nonselective nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester and 20 mg/kg of the selective inducible nitric oxide synthase inhibitor S-methylisothiourea. Animals were exposed to hyperbaric oxygen (2.8 atmospheres absolute for 90 minutes twice daily) with or without the administration of L-arginine and nitric oxide synthase inhibitors. RESULTS: Cyclophosphamide injection resulted in severe cystitis. S-methylisothiourea produced marked inhibition of cyclophosphamide induced bladder tissue damage. L-arginine and L-NG-nitroarginine methyl ester failed to a show meaningful protective effect. Hyperbaric oxygen protected the bladder only against ulceration. Moreover, hyperbaric oxygen did not contribute to the protective effects of L-arginine, L-NG-nitroarginine methyl ester or S-methylisothiourea. CONCLUSIONS: Nitric oxide produced by inducible nitric oxide synthase is an important mediator in the pathogenesis of cyclophosphamide induced cystitis. Hyperbaric oxygen has a beneficial effect on repairing bladder damage rather than on bladder protection.