Tempol Treatment Research Articles

Neuroprotective effect of tempol (4 hydroxy-tempo) on neuronal death induced by sciatic nerve transection in neonatal rats.

Peripheral nerve injury in newborn rats triggers extensive neuronal death within the spinal cord. Because most neurodegeneration is related to oxidative stress and apoptosis, the use of antioxidants may be of therapeutic interest. Tempol is promising because of its ability to chelate reactive oxygen species and to minimize or even prevent tissue damage. Here, we evaluated neuroprotective effects of tempol following neonatal sciatic nerve transection. Two-day-old pups underwent sciatic nerve axotomy followed by tempol (12, 24 and 48 mg/kg) treatment (i.p.) at 10 min, 6 h, and every 24 h up to 1 week after injury. The rats were then killed for lumbar intumescence analysis. Nissl staining, TUNEL, synaptophysin immunolabeling and qRT-PCR (Caspase 3, Bax and Bcl2) were carried out. The results indicated that tempol treatment, at 24 mg/kg, increased up to 21% spinal cord motoneuron survival (p<0.001), also preserving pre-synaptic terminals in the neuropile. Likewise, the TUNEL-positive cell number decreased in tempol-treated animals. qRT-PCR results indicated differential increase in Caspase 3 (3-fold), Bax (13-fold) and Bcl2 (28-fold) gene expression, after 12 h following axotomy and tempol treatment. In conclusion, tempol administration has proven to be neuroprotective after neonatal nerve injury, leading to improved motoneuron survival, synapse preservation and minimizing apoptosis.

Oxidative stress and autonomic dysregulation contribute to the acute time-dependent myocardial depressant effect of ethanol in conscious female rats.

The molecular mechanisms of the acute hypotensive and indirectly assessed cardiac depressant effect of ethanol (EtOH)-evoked myocardial depression and hypotension in female rats are not known. We tested the hypothesis that a time-dependent myocardial depression caused by EtOH is initiated by its direct and indirect (cardiac vagal dominance) effects and is exacerbated by gradual development of oxidative stress. In conscious female rats, we directly measured left ventricular developed pressure (LVDP), the maximal rise of ventricular pressure over time (dP/dtmax ), blood pressure (BP), heart rate (HR), and sympathovagal activity following intragastric EtOH (1 g/kg) or water over 90 minutes. Catalytic activity of acetaldehyde (ACA)-generating (alcohol dehydrogenase [ADH] and catalase) and eliminating aldehyde dehydrogenase [ALDH2] enzymes along with mediators of oxidative stress were measured in myocardial tissues collected at 30, 60, or 90 minutes after EtOH or water. EtOH reduced myocardial function (LVDP and dP/dtmax ) within 5 to 10 minutes before the steady fall in BP in conscious proestrus rats. Further, EtOH shifted the sympathovagal balance, analyzed by spectral analysis of high frequency and low frequency of interbeat intervals, toward vagal dominance. Prior vagal blockade (atropine) or antioxidant (tempol) treatment attenuated EtOH-evoked myocardial depression and hypotension. Ex vivo studies revealed time-dependent: (i) enhancement of ADH, but not ALDH2 activity (indicative of elevated ACA levels), (ii) increases in phosphorylated Akt and ERK1/2, NADPH-oxidase activity, reactive oxygen species, malondialdehyde, and 4-hydroxy-2-nonenal-modified proteins. These molecular responses along with reduced myocardial catalase activity were most evident at 90 minutes post-EtOH when the reductions in cardiac function and BP reached their nadir. Vagal dominance and time-dependent myocardial oxidative stress along with the accumulation of cardiotoxic aldehydes mediate EtOH-evoked myocardial dysfunction and hypotension in conscious proestrus female rats.

Hydrogen sulphide and tempol treatments improve the blood pressure and renal excretory responses in spontaneously hypertensive rats

Oxidative stress and suppressed H2S production lead to increased renal vascular resistance, disturbed glomerular hemodynamics, and abnormal renal sodium and water handling, contribute to the pathogenesis and maintenance of essential hypertension in man and the spontaneously hypertensive rat. This study investigated the impact of H2S and tempol alone and in combination on blood pressure and renal hemodynamics and excretory functions in the SHR. Groups of WKY rats or SHR (n = 6) were treated for 4 weeks either as controls or received NaHS (SHR + NaHS), tempol (SHR + Tempol), or NaHS plus tempol (SHR + NaHS + Tempol). Metabolic studies were performed on days 0, 14, and 28, thereafter animals were anaesthetized to measure renal hemodynamics and plasma oxidative and antioxidant markers. SHR control rats had higher mean arterial blood pressure (140.0 ± 2 vs. 100.0 ± 3 mmHg), lower plasma and urinary H2S, creatinine clearance, urine flow rate and urinary sodium excretion, and oxidative stress compared to WKY (all p < 0.05). Treatment either with NaHS or with tempol alone decreased blood pressure and oxidative stress and improved renal hemodynamic and excretory function compared to untreated SHR. Combined NaHS and tempol therapy in SHRs caused larger decreases in blood pressure (∼20–22% vs. ∼11–15% and ∼10–14%), increases in creatinine clearance, urinary sodium excretion and fractional sodium excretion and up-regulated the antioxidant status compared to each agent alone (all p < 0.05). These findings demonstrated that H2S and tempol together resulted in greater reductions in blood pressure and normalization of kidney function compared with either compound alone.

Acute superoxide radical scavenging reduces blood pressure but does not influence kidney function in hypertensive rats with postischemic kidney injury.

Acute kidney injury (AKI) is associated with significant morbidity and mortality in hypertensive surroundings. We investigated superoxide radical molecules influence on systemic haemodynamic and kidney function in spontaneously hypertensive rats (SHR) with induced postischemic AKI. Experiment was performed in anesthetized adult male SHR. The right kidney was removed, and left renal artery was subjected to ischemia by clamping for 40 minutes. The treated group received synthetic superoxide dismutase mimetic TEMPOL in the femoral vein 5 minutes before, during, and 175 minutes after the period of reperfusion, while the control AKI group received the vehicle via the same route. All parameters were measured 24 h after renal reperfusion. TEMPOL treatment significantly decreased mean arterial pressure and total peripheral resistance (P < 0.05) compared to AKI control. It also increased cardiac output and catalase activity (P < 0.05). Lipid peroxidation and renal vascular resistance were decreased in TEMPOL (P < 0.05). Plasma creatinine and kidney morphological parameters were unchanged among TEMPOL treated and control groups. Our study shows that superoxide radicals participate in haemodynamic control, but acute superoxide scavenging is ineffective in glomerular and tubular improvement, probably due to hypertension-induced strong endothelial dysfunction which neutralizes beneficial effects of O2 − scavenging.

Nephroprotective effects of TVP1022, a non-MAO inhibitor S-isomer of rasagiline, in an experimental model of diabetic renal ischemic injury

Ischemic acute kidney injury (iAKI) in diabetes mellitus is associated with a rapid deterioration of kidney function, more than in nondiabetic subjects. TVP1022, a non-MAO inhibitor S-isomer of rasagiline, possesses antioxidative and antiapoptotic activities. The current study examines the effects of TVP1022 and tempol on iAKI in diabetic rats. Diabetes was induced by streptozotocin. iAKI was induced by clamping the left renal artery for 30 min in both diabetic and nondiabetic rats. The right intact kidney served as a control. Forty-eight hours following ischemia, urinary flow (V), sodium excretion (UNaV), and glomerular filtration rate (GFR) in both ischemic and nonischemic kidneys were determined. The nephroprotective effects of tempol and TVP1022 were examined in these rats. Hematoxylin and eosin staining, 4-hydroxynonenal (4-HNE) immunofluorescence, and nitrotyrosine immunohistochemistry were performed on renal tissues of the various experimental groups. Compared with normoglycemic rats, iAKI in diabetic animals caused more profound reductions in V, UNaV, and GFR. Tempol and TVP1022 treatment increased GFR two- and four-fold in diabetic ischemic kidney, respectively. Besides hemodynamic perturbations, iAKI markedly increased renal immunoreactive 4-HNE and nitrotyrosine staining in both diabetic and nondiabetic rats. Moreover, iAKI increased medullary necrosis, congestion, and casts. Noteworthy, these increases were to a larger extent in ischemic diabetic kidneys. TVP1022, and to a lesser extent tempol, decreased nitrotyrosine and 4-HNE immunoreactivities and necrosis and cast formation in the renal medulla. TVP1022 treatment improves renal dysfunction and histological changes in an iAKI diabetic model and suggests a role for TVP1022 therapy in kidney injury.

Aggravation of diabetic nephropathy in BCL-2 interacting cell death suppressor (BIS)-haploinsufficient mice together with impaired induction of superoxide dismutase (SOD) activity

B cell CLL/lymphoma 2 (BCL-2)-interacting cell death suppressor (BIS), known as an anti-stress and anti-apoptotic protein, has been reported to modulate susceptibility to oxidative stress. This study investigated the potential role of BIS as an antioxidant protein in diabetic nephropathy. Diabetes was induced in BIS-heterozygote (BIS-HT) mice via streptozotocin injections and the resulting phenotypes were compared with those of BIS-wild-type (BIS-WT) mice over the 20 weeks following diabetes induction. Renal injuries, represented by increased plasma creatinine levels and increased albuminuria, were greater in diabetic BIS-HT mice than in diabetic BIS-WT mice, and were accompanied by a significant increase in reactive oxygen species (ROS) and oxidative stress markers. Moreover, renal pathological changes and the apoptotic process were accelerated in diabetic BIS-HT mice compared with diabetic BIS-WT mice with the same degree of hyperglycaemia; all were restored by 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol) treatment. The levels of NADPH oxidase and related proteins were not significantly higher in diabetic BIS-HT mice compared with diabetic BIS-WT mice. However, levels of superoxide dismutase (SOD)1 and SOD2 increased on the induction of diabetes in BIS-WT mice but not in BIS-HT mice, correlating with the total SOD activity. An in vitro study showed that knockdown of BIS production also resulted in impaired induction of SOD activity as well as SOD levels in HK-2 and NMS cells, concomitant with significant ROS accumulation. Our results suggest that the decreased antioxidant capacity of BIS aggravates diabetic nephropathy in diabetic BIS-HT mice, possibly as a result of the disruption in the regulation of SOD protein quality under oxidative stress.

Nox2-Derived Superoxide Contributes to Cerebral Vascular Dysfunction in Diet-Induced Obesity

Obesity is an increasing epidemic worldwide; however, little is known about effects of obesity produced by high-fat diet (HFD) on the cerebral circulation. The purpose of this study was to examine the functional and temporal effects of a HFD on carotid and cerebral vascular function and to identify mechanisms that contribute to such functional alterations. Responses of cerebral arterioles (in vivo) and carotid arteries (in vitro) were examined in C57Bl/6 (wild-type) and Nox2-deficient (Nox2(-/-)) mice fed a control (10%) or a HFD (45% or 60% kcal of fat) for 8, 12, 30, or 36 weeks. In wild-type mice, a HFD produced obesity and endothelial dysfunction by 12 and 36 weeks in cerebral arterioles and carotid arteries, respectively. Endothelial function could be significantly improved with Tempol (a superoxide scavenger) treatment in wild-type mice fed a HFD. Despite producing a similar degree of obesity in both wild-type and Nox2(-/-) mice, endothelial dysfunction was observed only in wild-type, but not in Nox2(-/-), mice fed a HFD. Endothelial dysfunction produced by a HFD occurs in a temporal manner and appears much earlier in cerebral arterioles than in carotid arteries. Genetic studies revealed that Nox2-derived superoxide plays a major role in endothelial dysfunction produced by a HFD. Such functional changes may serve to predispose blood vessels to reduced vasodilator responses and thus may contribute to alterations in cerebral blood flow associated with obesity.

Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity

The antioxidant tempol reduces obesity in mice. Here we show that tempol alters the gut microbiome by preferentially reducing the genus Lactobacillus and its bile salt hydrolase (BSH) activity leading to the accumulation of intestinal tauro-β-muricholic acid (T-β-MCA). T-β-MCA is an farnesoid X receptor (FXR) nuclear receptor antagonist, which is involved in the regulation of bile acid, lipid and glucose metabolism. Its increased levels during tempol treatment inhibit FXR signalling in the intestine. High-fat diet-fed intestine-specific Fxr-null (Fxr(ΔIE)) mice show lower diet-induced obesity, similar to tempol-treated wild-type mice. Further, tempol treatment does not decrease weight gain in Fxr(ΔIE) mice, suggesting that the intestinal FXR mediates the anti-obesity effects of tempol. These studies demonstrate a biochemical link between the microbiome, nuclear receptor signalling and metabolic disorders, and suggest that inhibition of FXR in the intestine could be a target for anti-obesity drugs.

The oxygen free radicals control MMP-9 and transcription factors expression in the spontaneously hypertensive rat

Oxygen free radical and matrix metalloproteinases-9 (MMP-9) play an important pathophysiological role in the development of chronic hypertension. MMP-9 activities are regulated at different levels. We hypothesize that as mediators of the expression of MMP-9 the transcription factors like nuclear factor kappa B (NF-κB), c-fos and retinoic acid receptors-α (RAR-α) with binding sites to the MMP-9 promoter are overexpressed in the spontaneously hypertensive rat (SHR) in a process that is regulated by oxygen free radicals. Transcription factor NF-κB, c-fos and RAR-α expression levels were determined by immunohistochemistry in renal, cardiac and mesentery microcirculation of the SHR and its normotensive control, the Wistar Kyoto (WKY) rat. The animals were treated with a superoxide scavenger (Tempol) for eight weeks. The elevated plasma levels of thiobarbituric acid reactive substances and MMP-9 levels in the SHR were significantly decreased by Tempol treatment (P<0.05). The NF-κB, c-fos and RAR-α expression levels in renal glomerular, heart and mesentery microvessels were enhanced in the SHR and could also be reduced by Tempol compared to untreated animals (P<0.05). The enhanced MMP-9 levels in SHR microvessels co-express with transcription factors. These results suggest that elevated NF-κB, c-fos and RAR-α expressions and MMP-9 activity in the SHR are superoxide-dependent.

Centrally Mediated Erectile Dysfunction in Rats with Type 1 Diabetes: Role of Angiotensin II and Superoxide

Erectile dysfunction is a serious complication of diabetes mellitus. Apart from the peripheral actions, central mechanisms are also responsible for penile erection. This study aims to determine the contribution of angiotensin (ANG) II in the dysfunction of central N-methyl-D-aspartic acid (NMDA)- and nitric oxide (NO)-induced erectile responses in streptozotocin-induced type 1 diabetic (T1D) rats. Three weeks after streptozotocin injections, rats were randomly treated with the angiotensin-converting enzyme inhibitor-enalapril, or the ANG II type 1 receptor blocker, losartan, or the superoxide dismutase mimetic, tempol, or vehicle via chronic intracerebroventricular infusion by osmotic mini-pump for 2 weeks. Central NMDA receptor stimulation or the administration of the NO donor, sodium nitroprusside (SNP)-induced penile erectile responses and concurrent behavioral responses were monitored in conscious rats. Two weeks of enalapril, losartan, or tempol treatment significantly improved the erectile responses to central microinjection of both NMDA and SNP in the paraventricular nucleus (PVN) of conscious T1D rats (NMDA responses-T1D+enalapril: 1.7 ± 0.6, T1D+losartan: 2.0 ± 0.3, T1D+tempol: 2.0 ± 0.6 vs. T1D+vehicle: 0.6 ± 0.3 penile erections/rat in the first 20 minutes, P < 0.05; SNP responses-T1D+enalapril: 0.9 ± 0.3, T1D+losartan: 1.3 ± 0.3, T1D+tempol: 1.4 ± 0.4 vs. T1D+vehicle: 0.4 ± 0.2 penile erections/rat in the first 20 minutes, P < 0.05). Concurrent behavioral responses including yawning and stretching, induced by central NMDA and SNP microinjections, were also significantly increased in T1D rats after enalapril, losartan, or tempol treatments. Neuronal NO synthase expression within the PVN was also significantly increased, and superoxide production was reduced in T1D rats after these treatments. These data strongly support the contention that enhanced ANG II mechanism/s within the PVN of T1D rats contributes to the dysfunction of central NMDA-induced erectile responses in T1D rats via stimulation of superoxide.

Tempol ameliorates cardiac fibrosis in streptozotocin-induced diabetic rats: role of oxidative stress in diabetic cardiomyopathy

Long-standing diabetes is associated with increased oxidative stress and cardiac fibrosis. This, in turn, contributes to the progression of cardiomyopathy. The present study was sought to investigate whether the free radical scavenger, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol) can protect against diabetic cardiomyopathy and to explore the specific underlying mechanism(s) in this setting. Diabetes was induced in rats by a single intraperitoneal injection dose of streptozotocin (50 mg/kg). These animals were treated with tempol (18 mg kg(-1) day(-1), orally) for 8 weeks. Our results showed significant increases in collagen IV and fibronectin protein levels and a marked decrease in matrix metalloproteinase-2 (MMP-2) activity measured by gelatin-gel zymography alongside elevated cardiac transforming growth factor (TGF)-β level determined using ELISA or immunohistochemistry in cardiac tissues of diabetic rats compared with control. This was accompanied by an increased in the oxidative stress as evidenced by increased reactive oxygen species (ROS) production and decreased antioxidant enzyme capacity along with elevated lactate dehydrogenase (LDH) and creatine kinase (CK-MB) serum levels as compared with the control. Tempol treatment significantly corrected the changes in the cardiac extracellular matrix, TGF-β, ROS or serum LDH, CK-MB levels, and normalized MMP-2 activity along with preservation of cardiac tissues integrity of diabetic rats against damaging responses. Moreover, tempol normalized the elevated systolic blood pressure and improved some cardiac functions in diabetic rats. Collectively, our data suggest a potential protective role of tempol against diabetes-associated cardiac fibrosis in rats via reducing oxidative stress and extracellular matrix remodeling.

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (Nω-nitro-L-arginine methyl ester; 100mg/kg; po)-induced hypertension treated with TEMPOL (18mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.

The effects of tempol on renal function and hemodynamics in cyclosporine-induced renal insufficiency rats

This study investigated the effects of tempol, a superoxide dismutase (SOD) mimetic and L-NAME, a nitric oxide (NO) synthase inhibitor on the renal function and hemodynamics in cyclosporine A (CsA) induced renal insufficiency rats. Male Sprague–Dawley rats were treated with either vehicle (C), tempol (T, 1 mmol/L in drinking fluid), L-NAME (L, 1 mmol/L in drinking fluid), CsA (Cs, 25 mg/kg/day via gavage), CsA plus tempol (TCs), CsA plus L-NAME (LCs) or CsA plus a combination of tempol and L-NAME (TLCs) for 21 consecutive days. At the end of treatment regimen, the renal responses to noradrenaline (NA), phenylephrine (PE), methoxamine and angiotensin II (Ang II) were determined. Cs and LCs rats had lower creatinine clearance (0.7 ± 0.1 and 0.6 ± 0.5 vs. 1.3 ± 0.2 mL/min/kg) and fractional excretion of sodium (0.12 ± 0.02 and 0.17 ± 0.01 vs. 0.67 ± 0.04%) but higher systolic blood pressure (145 ± 2 and 178 ± 4 vs. 116 ± 2) compared to the control (all p < 0.05), respectively. Tempol treatment in TCs or TLCs prevented the increase in blood pressure and improved creatinine clearance and sodium excretion compared to untreated Cs. The renal vasoconstriction in Cs or LCs to NA, PE and Ang II were lower than control by ∼35–48% (all p < 0.05). In TCs or TLCs, there was enhanced renal vasoconstriction to all agonist by ∼39–114% compared to Cs. SOD is important to counterbalance the hypertensive effect of a defective NO system and to allow the normal vasoconstrictor response of the renal vasculature to adrenergic agonists and Ang II in a model of CsA-induced renal insufficiency.

Effect of free radical scavenger on c-jun activation in rats with crush syndrome

To evaluate the relationship between reactive oxygen species (ROS)-mediated kidney injuries and Jun N-terminal kinase (JNK) activity and the therapeutic effects of tempol in crush syndrome (CS) model rats. Male Wister rats were randomly divided into sham operation group (SOG), CS groups (CS6G, CS12G and CS24G) and tempol treatment group (TG, a ROS scavenger). CS model rats were established by crushing the hind limbs of rats with 15 kg pressure for 6 hours, and inferior caval vein blood and kidney samples were harvested at 6, 12, 24 hours after removing crush pressure. In TG, 100 mg/kg tempol was intraperitoneally injected into CS model rats after withdraw of crush pressure. In SOG, rats were fixed on the board without any crush pressure. The activation of c-jun was determined by western blotting. Serological parameters and the content of malondialdehyde (MDA) in kidney tissues were determined by standard methods. Acute kidney injury reached the peak at 12 hours after the crush pressure. Compared with SOG, the content of phosphorylated c-jun was significantly higher in CSG and TG (p < 0.05), and the content of phosphorylated c-jun in the CSG was significantly higher than that in TG (p < 0.05). Interestingly, the changes of the MDA content in the kidney tissues of the 3 groups were similar to the changes of phosphorylated c-jun content. ROS-mediated phosphorylation of c-jun may play important roles in the acute kidney injury of CS rats. Tempol can inhibit the phosphorylation of c-jun and alleviate the acute kidney injury.

Chronic antioxidant therapy lowers blood pressure in adult but not in young Dahl salt hypertensive rats: the role of sympathetic nervous system

It is well-known that salt hypertension is associated with increased oxidative stress. Since the development of salt hypertension is age-dependent, we were interested whether young and adult salt hypertensive Dahl rats differ in oxidative stress level and/or in the effects of chronic antioxidant therapy on blood pressure (BP) level and on the participation of particular vasoconstrictor/vasodilator systems in BP maintenance. Young (5-week-old) and adult (12-week-old) salt-sensitive (Dahl-S) male rats were fed high-salt diet (5% NaCl) and drank tempol solution (2 mm) for 5 weeks. BP was monitored with radiotelemetry and vasoconstrictor/vasodilator balance was evaluated at the end of experiment. Moreover, NO synthase activity, superoxide production and lipoperoxidation were determined in heart, kidney and aorta in separate subgroups of Dahl rats. Tempol treatment had quite opposite BP effects in young and adult Dahl-S rats. While it tended to increase BP in young salt hypertensive Dahl-S rats, it significantly lowered BP in the adult ones due to reduced sympathetic vasoconstriction. Importantly, high salt intake substantially reduced NO synthase activity in heart and kidney, and markedly increased superoxide production in kidneys and aorta of adult Dahl-S rats in which BP correlated positively with superoxide production in thoracic aorta and lipoperoxidation in kidneys. Chronic antioxidant therapy lowered BP only in adult salt hypertensive Dahl-S rats in which superoxide levels were increased in both kidneys and aorta. Blood pressure reduction induced by chronic tempol treatment is related to attenuated sympathetic vasoconstriction rather than to augmented NO-dependent vasodilatation.

Tempol prevents cardiac oxidative damage and left ventricular dysfunction in the PPAR-α KO mouse

Peroxisome proliferator-activated receptor (PPAR)-α deletion induces a profound decrease in MnSOD activity, leading to oxidative stress and left ventricular (LV) dysfunction. We tested the hypothesis that treatment of PPAR-α knockout (KO) mice with the SOD mimetic tempol prevents the heart from pathological remodelling and preserves LV function. Twenty PPAR-α KO mice and 20 age-matched wild-type mice were randomly treated for 8 wk with vehicle or tempol in the drinking water. LV contractile parameters were determined both in vivo using echocardiography and ex vivo using papillary muscle mechanics. Translational and posttranslational modifications of myosin heavy chain protein as well as the expression and activity of major antioxidant enzymes were measured. Tempol treatment did not affect LV function in wild-type mice; however, in PPAR-α KO mice, tempol prevented the decrease in LV ejection fraction and restored the contractile parameters of papillary muscle, including maximum shortening velocity, maximum extent of shortening, and total tension. Moreover, compared with untreated PPAR-α KO mice, myosin heavy chain tyrosine nitration and anion superoxide production were markedly reduced in PPAR-α KO mice after treatment. Tempol also significantly increased glutathione peroxidase and glutathione reductase activities (~ 50%) in PPAR-α KO mice. In conclusion, these findings demonstrate that treatment with the SOD mimetic tempol can prevent cardiac dysfunction in PPAR-α KO mice by reducing the oxidation of contractile proteins. In addition, we show that the beneficial effects of tempol in PPAR-α KO mice involve activation of the glutathione peroxidase/glutathione reductase system.

Effects of tempol on endothelial and vascular dysfunctions and insulin resistance induced by a high-fat high-sucrose diet in the rat

We investigated the effects of treatment with tempol (an antioxidant) on vascular and metabolic dysfunction induced by a high-fat high-sucrose (HFHS) diet. Rats were randomized to receive an HFHS or chow diet with or without tempol treatment (1.5 mmol·(kg body mass)(-1)·day(-1)) for 4 weeks. Blood pressure, heart rate, and blood flow were measured in the rats by using intravascular catheters and Doppler flow probes. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic-hyperinsulinemic clamp. In-vitro studies were performed to evaluate vascular reactivity and endothelial and inducible nitric oxide synthase (eNOS; iNOS) expression in vascular and muscle tissues. Endothelin, nitrotyrosine, and NAD(P)H oxidase expressions were determined in vascular tissues, and glucose transport activity and glucose transporter 4 (GLUT4) expression were examined in muscles. Tempol treatment was found to prevent alterations in insulin sensitivity, glucose transport activity, GLUT4 expression, and vascular reactivity, and to prevent increases in plasma insulin, blood pressure, and heart rate noted in the untreated HFHS-fed rats. These were associated with increased levels of eNOS expression in vascular and muscle tissues, but reductions in nitrotyrosine, endothelin, NAD(P)H oxidase, and iNOS expressions. Therefore, oxidative stress induced by a relatively short-term HFHS diet could contribute to the early development of vascular and metabolic abnormalities in rats.

Spreading vasodilatation in the murine microcirculation: attenuation by oxidative stress‐induced change in electromechanical coupling

Regulation of blood flow in microcirculatory networks depends on spread of local vasodilatation to encompass upstream arteries; a process mediated by endothelial conduction of hyperpolarization. Given that endothelial coupling is reduced in hypertension, we used hypertensive Cx40ko mice, in which endothelial coupling is attenuated, to investigate the contribution of the renin-angiotensin system and reduced endothelial cell coupling to conducted vasodilatation of cremaster arterioles in vivo. When the endothelium was disrupted by light dye treatment, conducted vasodilatation, following ionophoresis of acetylcholine, was abolished beyond the site of endothelial damage. In the absence of Cx40, sparse immunohistochemical staining was found for Cx37 in the endothelium, and endothelial, myoendothelial and smooth muscle gap junctions were identified by electron microscopy. Hyperpolarization decayed more rapidly in arterioles from Cx40ko than wild-type mice. This was accompanied by a shift in the threshold potential defining the linear relationship between voltage and diameter, increased T-type calcium channel expression and increased contribution of T-type (3 μmol l(-1) NNC 55-0396), relative to L-type (1 μmol l(-1) nifedipine), channels to vascular tone. The change in electromechanical coupling was reversed by inhibition of the renin-angiotensin system (candesartan, 1.0 mg kg(-1) day(-1) for 2 weeks) or by acute treatment with the superoxide scavenger tempol (1 mmol l(-1)). Candesartan and tempol treatments also significantly improved conducted vasodilatation. We conclude that conducted vasodilatation in Cx40ko mice requires the endothelium, and attenuation results from both a reduction in endothelial coupling and an angiotensin II-induced increase in oxidative stress. We suggest that during cardiovascular disease, the ability of microvascular networks to maintain tissue integrity may be compromised due to oxidative stress-induced changes in electromechanical coupling.

Outcome of Periapical Lesions in a Rat Model of Type 2 Diabetes: Refractoriness to Systemic Antioxidant Therapy

IntroductionThis study evaluated the development of periapical lesions in a rat model of type 2 diabetes and assessed the potential actions of the antioxidant agent tempol in this model. MethodsMale Wistar rats were used; they received tap water (N = 5) or a 20% glucose solution (N = 15) during a period of 9 weeks. At the sixth week, periapical lesions were induced on the first mandibular molars, and the animals were subdivided into 4 groups. The subgroup 1 was composed of nondiabetic rats orally receiving saline solution (10 mL/kg). Chronically glucose-fed rats were divided into the following subgroups: (2) saline-treated animals (10 mL/kg by oral route), and animals treated with tempol by gavage at doses of (3) 50 mg/kg or (4) 100 mg/kg. The body weight was monitored thoroughly. After 21 days of apical periodontitis induction, the animals were killed, and the mandibles were collected and submitted to radiographic and histologic analysis. The livers were collected to determine free radicals, and the blood plasma was used to measure insulin levels. ResultsType 2 diabetic rats displayed a significant decrease of body weight gain and a slight increase of insulin levels, which were allied to reduced levels of the antioxidant components catalase and reduced glutathione; these alterations were reversed by tempol. Concerning the periapical lesions, neither radiographic nor histologic analysis revealed any significant difference between control and type 2 diabetic rats. In diabetic rats, the apical periodontitis was refractory to tempol treatment. ConclusionsThe extent and cellularity of periapical lesions in glucose-fed type 2 diabetic rats were similar to those seen in control rats. Despite affecting other parameters related to diabetes, tempol failed to improve the outcome of endodontic lesions in type 2 diabetic animals.

Impact of chronic intermittent hypoxia upon rat liver lipid metabolism and interventional effect of Tempol

To explore the impact of chronic intermittent hypoxia (CIH) upon rat liver lipid metabolism and effect of anti-oxidant Tempol. Male Wistar rats (n = 80) were randomly divided into intermittent hypoxia group (10, 20, 30, 40 times/h), intermittent hypoxia Tempol treatment group, intermittent hypoxia normal saline treatment group, intermittent air mimic group (IA) and blank control group (CG). Sections of liver were stained with hematoxylin and eosin. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Levels of liver homogenate triglyceride (TG), total cholesterol (TC), free fatty acids (FFA) and serum TG, TC, adiponectin (ADP) were measured. Liver histology: IH group exhibited hepatocellular swelling, hyperchromatosis, disrupted hepatocellular membrane. With the increase of frequency, there were local necrosis and infiltration of inflammatory cells. But no steatosis was seen. Tempol early treatment and IA groups exhibited no hepatocellular swelling or inflammatory cell infiltration. The activities of ALT and AST increased along with the increased frequency in IH group (all P < 0.01). The levels of ALT and AST in IH group ((48.6 ± 3.6), (25.4 ± 2.6) U/L) were higher than those in IA group ((20.3 ± 3.1), (18.7 ± 1.3) U/L) and CG group ((17.5 ± 2.4), (18.8 ± 1.3) U/L) (all P < 0.01). It decreased in Tempol treatment group, and more obviously when early intervention was applied (all P < 0.01). Liver homogenate TG, TC and FFA had no difference among IH, IA and CG groups (all P > 0.05), and no difference in different frequencies in IH group (all P > 0.05). The levels of serum TG, TC in IH groups were higher than those in IA and CG groups while ADP was lower (all P < 0.01). It changed more obviously in different frequencies in IH group (all P < 0.01). In Tempol treatment group, serum TG, TC decreased while ADP increased and changed more obviously when early intervention was applied (all P < 0.01). CIH causes the morphologic changes of liver and the elevations of ALT and AST, but results not in lipid deposition in liver cells. Anti-oxidation of Tempol can block intermittent hypoxia associated with liver injury.