mmHg In Rats Research Articles

Influence of excessive salt consumption on the functional state of microvascular vessels of the skin of rats with renal dysfunction

Background. Cardiovascular complications are common in chronic kidney disease (CKD). The relationship between the cardiovascular system and the kidneys is complex and diverse, but the question of the mechanisms of the influence of excess NaCl consumption on the functional state of blood vessels during renal dysfunction remains relevant.The aim: to evaluate the effect of a high-salt diet on the functional state of microcirculatory vessels (MCR) of the skin in rats at an early stage of renal dysfunction.Material and Methods. The study was conducted on Wistar rats. The first SO group included animals subjected to sham surgery (SO) and receiving a standard diet (0.34% NaCl); in the second, HS group – animals that also underwent a sham operation, but received a high-salt diet (4% NaCl); in the third, NE group – rats subjected to ¾ nephrectomy (NE) and receiving a standard diet; the fourth, HS+ NE group included rats subjected to ¾ NE and fed a high-salt diet (4% NaCl). Blood pressure (BP) was measured in the tail using the cuff method (Sistola, Russia), MCR was assessed using laser Doppler flowmetry (LDF). The reactivity of skin microvessels was assessed by changes in the MCR index before and after iontophoresis of acetylcholine (ACh) and sodium nitroprusside (NP). Based on the assessment of the spectral characteristics of fluctuations in the MCR index obtained using Wavelet analysis, the value of endothelial, neurogenic and myogenic microvascular tone was calculated.Results. 4 months after ¾ NE, rats showed a decrease in the excretory function of the kidneys. In rats on a highsalt diet, an increase in blood urea levels was noted. The blood pressure in rats of the SO+HS group did not significantly differ from the control animals of the SO group (131 ± 8 and 125 ± 4 mmHg, respectively, NS), NE led to a significant increase in blood pressure (135 ± 5 mmHg in rats of the NE group, and 145 ± 7 mmHg – in the NE+HS group). The average MCR was higher in rats receiving a high-salt diet (SO+HS and NE+HS groups), compared with animals of the corresponding group receiving a standard diet. The combination of NE and high-salt load has an inhibitory effect on the amplitude of fluctuations in the intensity of perfusion in all studied ranges: endothelial, neurogenic and myogenic, while the calculated value of skin microvascular tone in three ranges was the highest in NE+HS group rats. In all experimental groups, the reactivity to AH was reduced compared with the SO group, and after NP iontophoresis, the average perfusion rate significantly decreased only in groups of animals receiving a high-salt diet: SO+HS and NE+HS.Conclusion. A high-salt diet in rats with an initial stage of renal dysfunction contributes to an increase in blood pressure and a decrease in natriuresis compared with animals with NE who received a standard diet. With excessive consumption of table salt in rats after NE, the intensity of cutaneous MCR increases, while reducing the variability of the MCR index. A high-salt diet in rats with NE modifies the characteristics of the spectral components of fluctuations in the MCR index, indicating an increase in tonic effects on skin microvessels in the endothelial, neurogenic and myogenic ranges. With hypernatrial load, animals with ¾ NE have a decrease in the reactivity of cutaneous microvessels to AH and NP, which is mediated by both a decrease in endothelial NO production and a decrease in MMC sensitivity to NO.

Large-Volume Intrathecal Administrations: Impact on CSF Pressure and Safety Implications.

The increasing number of studies demonstrates the high potency of the intrathecal (IT) route for the delivery of biopharmaceuticals to the central nervous system (CNS). Our earlier data exhibited that both the infused volume and the infusion rate can regulate the initial disposition of the administered solute within the cerebrospinal fluid (CSF). This disposition is one of key factors in defining the subsequent transport of the solute to its intended target. On the other hand, fast additions of large volumes of liquid to the CSF inevitably raise the CSF pressure [a.k.a. intracranial pressure (ICP)], which may in turn lead to adverse reactions if the physiologically delimited threshold is exceeded. While long-term biological effects of elevated ICP (hydrocephalus) are known, the safety thresholds pertaining to short-term ICP elevations caused by IT administrations have not yet been characterized. This study aimed to investigate the dynamics of ICP in rats and non-human primates (NHPs) with respect to IT infusion rates and volumes. The safety regimes were estimated and analyzed across species to facilitate the development of translational large-volume IT therapies. The data revealed that the addition of a liquid to the CSF raised the ICP in a rate and volume-dependent manner. At low infusion rates (<0.12 ml/min in rats and <2 ml/min in NHPs), NHPs and rats displayed similar tolerance patterns. Specifically, safe accommodations of such added volumes were mainly facilitated by the accelerated pressure-dependent CSF drainage into the blood, with I stabilizing at different levels below the safety threshold of 28 ± 4 mm Hg in rats and 50 ± 5 mm Hg in NHPs. These ICPs were safely tolerated for extended durations (of at least 2–25 min). High infusion rates (including boluses) caused uncompensated exponential ICP elevations rapidly exceeding the safety thresholds. Their tolerance was species-dependent and was facilitated by the compensatory role of the varied components of craniospinal compliance while not excluding the possibility of other contributing factors. In conclusion, large volumes of liquids can safely be delivered via IT routes provided that ICP is monitored as a safety factor and cross-species physiological differences are accounted for.

Abstract 036: Genetics and Epigenetics on Circadian Blood Pressure and Variability in Rats Compared With Ambulatory Blood Pressure Variability in Human

Hypertension is associated with high morbidity and mortality. Blood pressure (BP) levels vary widely due to predictable biological rhythms and unpredictable environmental factors. Daily (circadian) rhythm characteristics are considered essential parameters for recognizing and treating increased risks in BP. Franz Halberg spent most of his academic career in cardiovascular research, focused on ambulatory monitoring and developing chronobiological methods for clinical application. To compare BP Variability (BPV), automatically monitored ambulatory BP around the clock at 30-60 min intervals in 20 human subjects was compared with telemetered circadian BP in rats after one-cell homozygous embryo-transfer into spontaneously hypertensive (SHR, pup:shr) or normotensive (WKY, pup:wky) rats' oviducts (embryos: s,w; oviduct-uterus: S,W) and cross-suckled at birth (nurses S,W). The circadian response peaked in the late afternoon hours in most human subjects and early morning hours in rats. Human circadian double amplitudes (2A) varied from 8 to 26 mm Hg with higher 2A in elder adults, and 3-8 mm Hg in rats with significantly higher fluctuations in SHR groups (7.5±0.7 for sSS, 8.3±0.6 for sSW vs. 4.7±0.3 mm Hg for wWW). The circaseptan 2A in the adolescents were 10±1 for SBP and 12±3 mm Hg for DBP, and sharp increased winter 2A (SBP 54; HR 48, both P&lt;0.0001) were observed. In rats, mostly clustered peaks around the light-on hours, while sWS and wSS groups showed peak-hour variations (19:18 to 08:06 and 02:58 to 09:10 hrs, respectively). The peak-hour in shr strains was shifted to earlier or delayed in the WKY uterus (sWW and sWS groups) with large deviations as compared to the wWW group. Hypertensive-prone shr strains showed significantly lowered BPs in WKY uterine and/or WKY nursing environments indicating that environment can strongly modify genetic influence, yet the lowered shr MESORs by the WKY environments remained above the MESORs encountered in wky donors. Thus, the maternal environment may be a major factor that affects the offspring’s BP and BPV later in life. Chronomes broader than circadian BPs are considered as a gauge of vascular disease status. Abnormal BP/BPV modifications may lead to novel implications in preventive and therapeutic strategies in CVD.

High salt diet impairs cerebral blood flow regulation via salt-induced angiotensin II suppression.

This study sought to determine whether salt-induced ANG II suppression contributes to impaired CBF autoregulation. Cerebral autoregulation was evaluated with LDF during graded reductions of blood pressure. Autoregulatory responses in rats fed HS (4% NaCl) diet vs LS (0.4% NaCl) diet were analyzed using linear regression analysis, model-free analysis, and a mechanistic theoretical model of blood flow through cerebral arterioles. Autoregulation was intact in LS-fed animals as MAP was reduced via graded hemorrhage to approximately 50mm Hg. Short-term (3days) and chronic (4weeks) HS diet impaired CBF autoregulation, as evidenced by progressive reductions of laser Doppler flux with arterial pressure reduction. Chronic low dose ANG II infusion (5mg/kg/min, i.v.) restored CBF autoregulation between the pre-hemorrhage MAP and 50mm Hg in rats fed short-term HS diet. Mechanistic-based model analysis showed a reduced myogenic response and reduced baseline VSM tone with short-term HS diet, which was restored by ANG II infusion. Short-term and chronic HS diet lead to impaired autoregulation in the cerebral circulation, with salt-induced ANG II suppression as a major factor in the initiation of impaired CBF regulation.

Placental CD4+ T cells isolated from preeclamptic women cause preeclampsia-like symptoms in pregnant nude-athymic rats.

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with a proinflammatory profile compared to normal pregnancy (NP). We hypothesize that CD4+ T cells from PE patient placentas cause PE symptoms during pregnancy compared to those from NP women. CD4+ T cells were isolated from placentas of PE and NP women using anti-CD4 magnetic separation, cultured in TexMACS medium at 37 °C in 5% CO2, and injected intraperitoneally into nude-athymic rats on day 12 of gestation. On day 18, carotid catheters were implanted and on day 19, mean arterial pressure (MAP) was measured and blood and tissues were collected. MAP was 125 ± 2 mmHg in rats with NP CD4+ T cells but increased to 140 ± 4 mmHg in rats with PE CD4+ T cells. Significant differences in circulating cytokines tumor necrosis factor-alpha (TNF-α), interleukin-17 (IL-17) and soluble fms-like tyrosine kinase-1 (sFlt-1) were found with PE vs NP CD4+ T cells (TNF-α- PE = 167.4 pg/mL, NP = 79.4 pg/mL; IL-17-PE = 7.054 pg/mL, NP = 3.185 pg/mL; sFlt-1-PE = 90.7 pg/mL, NP = 58.2 pg/mL. In addition, renal cortical endothelin-1 (ET-1) mRNA expression increased 4.5 fold in rats with PE CD4+ T cells versus those receiving to NP CD4+ T cells. These data indicate an important role for placental PE CD4+ T cells to cause many characteristics of PE during pregnancy.

Chronic renal artery insulin infusion increases mean arterial pressure in male Sprague-Dawley rats.

Hyperinsulinemia has been hypothesized to cause hypertension in obesity, type 2 diabetes, and metabolic syndrome through a renal mechanism. However, it has been challenging to isolate renal mechanisms in chronic experimental models due, in part, to technical difficulties. In this study, we tested the hypothesis that a renal mechanism underlies insulin hypertension. We developed a novel technique to permit continuous insulin infusion through the renal artery in conscious rats for 7 days. Mean arterial pressure increased by ~10 mmHg in rats that were infused intravenously (IV) with insulin and glucose. Renal artery doses were 20% of the intravenous doses and did not raise systemic insulin levels or cause differences in blood glucose. The increase in blood pressure was not different from the IV group. Mean arterial pressure did not change in vehicle-infused rats, and there were no differences in renal injury scoring due to the renal artery catheter. Glomerular filtration rate, plasma renin activity, and urinary sodium excretion did not differ between groups at baseline and did not change significantly with insulin infusion. Thus, by developing a novel approach for chronic, continuous renal artery insulin infusion, we provided new evidence that insulin causes hypertension in rats through actions initiated within the kidney.

Abstract P272: Preeclamptic Placental CD4+ T Cells Cause Preeclamptic Like Features in Normal Pregnant Nude Athymic Rats

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with a proinflammatory profile compared to normal pregnancy (NP). We hypothesize that CD4 + T cells play an important role to cause much of the pathophysiology associated with PE. To determine if CD4 + T cells isolated from PE patients cause PE symptoms during pregnancy compared to those collected from NP women, CD4 + T cells were isolated from placentas of both PE and NP women using magnetic separation with anti-CD4 antibodies, cultured in TexMACS medium with IL-2 at 37°C in 5% CO 2 , and injected intraperitoneally into pregnant, nude-athymic rats on day 12 of gestation. On day 18, carotid catheters were implanted and on day 19 MAP was measured and blood and tissues were collected. MAP was 125±3 mmHg in rats with NP T cells but increased to 140±9 mmHg in rats with PE T cells. Significant differences in circulating cytokines TNF-α, IL-6, IL-17 and sFlt-1 were found with PE vs NP CD4 + T cells (TNF-α- PE=142.4 pg/mL, NP=79.4 pg/mL; IL-6 - PE=311.6 pg/mL, NP=277.8 pg/mL; IL-17-PE= 7.054 pg/mL, NP=3.185 pg/mL; sFlt-1-PE=90.7 pg/mL, NP=58.2 pg/mL. However, there was no difference in Isoprostane levels or IL-2 between the two groups (Isoprostane-PE=3628 pg/mL, NP=3061 pg/mL; IL-2-PE=125 pg/mL, NP=125 pg/mL). ROS in tissues was measured using chemiluminescence and no difference was found in placenta while renal ROS showed an increased with PE CD4 + T cells vs NP CD4 + T cells (ROS-PE=5528 RLU/min/mg protein, NP=3372 RLU/min/mg protein). In addition, no difference in ET-1 mRNA was found in the placenta of nude rats receiving PE CD4+T cells, however, renal cortical ET-1 mRNA expression was increased 4.5 fold in rats with PE CD4+ T cells compared to those receiving to NP CD4+ T cells. These data indicate an important role for placental PE CD4 + T cells to cause inflammation and stimulate renal vasoactive pathways that may contribute to hypertension during pregnancy. This research is funded by R01HD067541-06

Dexmedetomidine effect to lung injury in abdominal hypertension

Objective: An experimental study was performed to evaluate the effect of dexmedetomidine on lung injury secondary to experimental intra-abdominal hypertension (IAH). Methods: Eighteen Wistar-albino rats were included and allocated into 3 groups - the control group (CG, n=6), the sham group (SG, n=6) and the dexmedetomidine group (DG, n=6). No intervention was made in CG. IAH was achieved by insufflating atmospheric air with a percutaneous intraperitoneal needle up to 15 mmHg pressures in SG and DG. At the 60th min of IAH, physiological serum (1.5 ml/100 grams/hr) in SG and dexmedetomidine (1 mcg/kg/hr) in DG were infused for 30 min through the tail vein. At the 90th min, the left inferior lobes of the lung were harvested for biochemical (nitric oxide-NO, malondialdehyde-MDA) and histopathological (alveolar hemorrhage, edema, congestion, leukocyte infiltration) examination. Results: There was no significant difference between any of the groups with regards to NO and MDA levels (p>0.05). His- topathologically, although alveolar hemorrhage, edema, congestion, leukocyte infiltration were increased in SG compared to CG and DG, the difference was not statistically significant (p>0.05). There was no significant difference between CG and DG with respect to histopathological grading (p>0.05). Conclusion: IAP of 15 mmHg in rats causes mild injury in lung parenchyma. The administration of DEX in clinical doses does not seem to significantly affect the lungs of rats.

Abstract 047: Genetics and Environmental Influences on Ambulatory Blood Pressure in Rats Compared With Circadian Blood Pressure Variability in Human

Genetic susceptibility is an important factor in raising blood pressure (BP). Daily (circadian) rhythm characteristics are considered essential parameters for recognizing and treating increased risks in BP. To examine BP in genetics with environmental modifications, one-cell homozygous embryos were transferred into spontaneously hypertensive (SHR, pup:shr) or normotensive (WKY, pup:wky) rats' oviducts (embryos: s, w; oviduct-uterine: S, W) in a reciprocal fashion. Pups were cross-suckled at birth (nurses S, W) and weaned to normal diets at day-21. At Day-120, telemetered BPs were monitored for 5 consecutive days every 4 min and analyzed by the method of ANOVA. Ambulatory BP in 20 adolescents and adults were monitored automatically around the clock at 30-min to hourly intervals and analyzed by the least square rhythmometry method. As expected, shr BPs were markedly reduced when they were transplanted to the W-uterine and/or the W-lactation milieu (sSS vs. sSW, sWS and sWW: 197 vs.178, 147 and 178 mm Hg). BP in wky was significantly altered only in the wSW group (wWW vs, wSW: 127 vs.131 mm Hg). All subjects showed significant circadian fluctuations with a peak in the late afternoon hours in most human subjects and rats as a nocturnal animal mostly close to midnight hours, while shr with W-uterine (sWS) a bit delayed peak hour (00:45) and with combined W-uterine/W-nursing (sWW) a bit earlier peak hour (20:12). Circadian double amplitudes (2A) in the human subjects varied from 8 to 26 mm Hg with higher 2A in elder adults, and 3-8 mm Hg in rats with significantly higher fluctuations in shr groups as compared to that of wWW (7.5±0.7 for sSS, 8.3±0.6 for sSW vs. 4.7±0.3 mm Hg for wWW). The hypertensive-prone shr strain showed significantly lowered BPs in a normotensive WKY uterine environment and/or by WKY nursing mothers, indicating that environment influences can strongly modify genetic factors, yet the lowered shr MESORs by the WKY environments remained above the MESORs encountered in wky donors. Chronomes broader than circadian should be considered in interpreting BP responses as a gauge of vascular disease status.

High-level expression of angiotensin converting enzyme inhibitory peptide Tuna AI as tandem multimer in Escherichia coli BL21 (DE3)

Food-derived angiotensin converting enzyme inhibitory peptides (ACEIP) show a great promise for prevention and treatment of hypertension. Tuna muscle derived peptide, Tuna AI (PTHIKWGD), is a potent ACEIP with high activity and stability. To improve the expression level of Tuna AI in Escherichia coli, multimer genes of Tuna AI were assembled and expressed as fusion proteins by constructing the vectors of pET30-4nTunaAI (n=1, 2, 4 and 8). The results showed the multimer genes were successfully expressed in the constructed system, and their expression level improved significantly with increasing repeat degree. The E. coli harboring pET30-32TunaAI was selected for production of peptide based on its highest expression level and its fusion protein was suit for urea-washing purification. The peptide monomer was released from the fusion protein by formic acid cleavage, and successively purified by membrane filtration, cation exchange chromatography and size exclusion chromatography, with a final yield of 218.9mg/L. The purified monomeric peptide was confirmed by mass spectrometry analysis, N-terminal sequencing, and activity measurement. This peptide resulted in a significant decrease of systolic blood pressure by 36.5mmHg in rats at 4h. These results suggest our strategy is an economic solution for the mass production of antihypertensive peptides.

Abstract 294: CD4 + T cells Isolated from Preeclamptic Women Produce a Preeclampsia-Like Phenotype in Pregnant Nude-Athymic Rats

Preeclampsia (PE) is the development of hypertension, with proteinuria, during the second half of pregnancy and is associated with an altered immune function, resulting in a chronic state of inflammation, which is believed to play an important role to increase vasoconstrictors such as Endothelin (ET-1) and autoantibodies as well as much of the pathophysiology associated with PE. It has been previously shown that women with PE have an altered CD4 + T cell population compared to normal pregnant women. In addition, Ana Zenclussen’s lab has shown that adoptive transfer of activated T helper 1 like splenocytes increased blood pressure, fetal rejection and cytokines secreted from uterine cells when transferred to normal pregnant mice. However, mechanisms causing the increased blood pressure and renal abnormalities were not further investigated. The purpose of this study was to determine if CD4 + T cells isolated from placentas of PE patients have altered consequences during pregnancy compared to those collected from normal pregnant (NP) women that may further define their role in causing the pathophysiology of PE. CD4 + T cells were isolated from placentas of PE and NP women, using magnetic anti-CD4 antibodies, cultured in TexMACS medium (Miltenyi Biotec) with IL-2 at 37°C in 5% CO 2 , and injected intraperitoneally into first timed pregnant, nude-athymic rats on day 12 of gestation. On day 18 carotid catheters were implanted and on day 19 MAP was measured and blood and tissues were collected. MAP was significantly increased from 125±3 mmHg in rats injected with NP CD4 + T cells to 140±9 mmHg in rats injected with PE CD4 + T cells. Circulating inflammatory cytokines IL-6 and TNF-α were measured and found to be increased in rats exposed to PE vs NP CD4 + T cells (TNF-α- PE=142.4 pg/mL, NP=79.4 pg/mL; IL-6 - PE=311.6 pg/mL, NP=277.8 pg/mL). Renal cortical ET-1 mRNA expression was found to be increased 4.5 fold in rats exposed to PE CD4+ T cells compared to those exposed to NP CD4+ T cells (p=0.06). These data suggest an important role for placental CD4 + T cells stimulated in women with PE to cause hypertension and inflammation and renal abnormalities during the course of this disease.

Estimating the clinical risk of hypertension from VEGF signal inhibitors by a non-clinical approach using telemetered rats

Anti-angiogenic drugs that target Vascular Endothelial Growth Factor (VEGF) signaling pathways caused hypertension as an adverse effect in clinical studies. Since the hypertension may limit the benefit provided for patients, the demand for non-clinical research that predicts the clinical risk of the hypertension has risen greatly. To clarify whether non-clinical research using rats can appropriately estimate the clinical risk of hypertension caused by VEGF signal inhibitors, we investigated the hemodynamic effects and pharmacokinetics (PK) of the VEGF signal inhibitors cediranib (0.1, 3, and 10 mg/kg), sunitinib (5, 10, and 40 mg/kg), and sorafenib (0.1, 1, and 5 mg/kg) in telemetered rats and examined the correlation between the non-clinical and the clinical hypertensive effect. The VEGF signal inhibitors significantly elevated blood pressure (BP) in rats within a few days of the initiation of dosing, and levels recovered after dosing ended. The trend of the hypertension was similar to that in clinical studies. We found that the AUC at which BP significantly increased by approximately 10 mmHg in rats was comparable to the clinical AUC at which moderate to severe hypertension occurred. These results represent correlations between the non-clinical and the clinical hypertensive effect of VEGF signal inhibitors, suggesting that non-clinical research using telemetered rats would be an effective approach to predict the clinical risk of hypertension caused by VEGF signal inhibitors.

Efficacy of centhaquin as a small volume resuscitative agent in severely hemorrhaged rats

Centhaquin has been reported to be an effective resuscitative agent. The present study was carried out to determine resuscitative effect of centhaquin when administered using a small volume of 3% hypertonic saline (HS) to hemorrhaged rats. Sprague-Dawley rats were anesthetized with urethane, and a pressure catheter SPR-320 was placed in the left femoral artery; another pressure-volume catheter SPR-869 was placed into the left ventricle. Hemorrhage was induced by withdrawing blood and mean arterial pressure (MAP) was maintained at 35 mm Hg for 30 minutes after which resuscitation was performed. Animals were divided in 2 groups: group A received HS and group B received centhaquin (0.05 mg/kg) dissolved in HS. The time by which MAP fell back to 35 mm Hg was observed at that time all animals were administered fresh blood. It was found that centhaquin significantly reduced blood lactate and improved cardiac output and MAP of hemorrhaged rats compared with HS. The time by which MAP fell back to 35 mm Hg in rats treated with HS was 55 ± 6 minutes, whereas it was 161 ± 14 minutes in centhaquin treated rats. Survival time following administration of fresh blood was 79 ± 7 minutes in vehicle-treated group, whereas it was 105 ± 9 minutes in centhaquin-treated rats. The total time of survival of rats treated with HS or centhaquin was 134 ± 12 minutes and 266 ± 16 minutes, respectively. Centhaquin, in small volume, maintained MAP of hemorrhaged rats for a considerable long time and improved the survival time.

Does prolonged pneumoperitoneum affect the kidney? Oxidative stress, stereological and electron microscopy study in a rat model

Pneumoperitoneum (Pp) at 12 to 15 mmHg in rats is associated with kidney damage. However, Pp at 8 mmHg is now known to best correlate to working pressures used in humans. Thus the aim of this work was to study the kidney of rats submitted to prolonged Pp at 8 mmHg. Rats were divided into a Sham group (n = 14), submitted to anesthesia, and a Pp group (n = 14), submitted to Pp at 8 mmHg, followed by deflation. In both groups, 7 animals were immediately killed and their kidneys were used for oxidative stress analyses. The remaining 7 rats in each group were evaluated after 6 weeks for the number of glomeruli and podocyte morphology. For all analyzed parameters Sham and Pp groups presented no statistical difference. When submitted to adequate Pp pressures (8 mmHg), no kidney damage occurs in rats.

Hyperoxia During Septic Shock—Dr. Jekyll or Mr. Hyde?

Enhanced oxidative and nitrosative stress resulting from increased formation of reactive oxygen (ROS) and reactive nitrogen (RNS) species is a common phenomenon during sepsis and referred to assume major importance during the development of shock-related hypotension, impairment of microcirculatory perfusion, mitochondrial dysfunction, and tissue injury (1). Because there is evidence in the literature that increasing the FIO2 leads to enhanced ROS production (2), current guidelines recommend that the least inspiratory O2 fraction (FIO2) be used that is associated with an arterial O2 tension (PaO2) of 55 to 80 mmHg or an arterial hemoglobin O2 saturation of 88% to 95%. In fact, the toxic effects of hyperoxia are well established for more than a century (2), the lung being particularly vulnerable to O2 toxicity, which presents as alveolitis, hyaline membranes and hemorrhagic pulmonary edema (2, 3). However, nothing is simple and easy: hyperoxia-related lung injury requires either prolonged exposure or the combination with “injurious ventilation” (3). Moreover, in various shock models of hemorrhagic shock (4) and ischemia/reperfusion injury (5, 6), pure O2 breathing reversed arterial hypotension (4, 5) and exerted anti-inflammatory (5) and antiapoptotic (6) properties, and these beneficial effects were confirmed during murine (7) and porcine fecal (3) peritonitis. It must be emphasized that both timing and dosing of hyperoxia seem to be crucial in this context: During murine zymosan-induced shock, pure O2 breathing improved survival, reduced hyperinflammation as documented by attenuated cytokine release, and increased the activity of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. However, the therapeutic effects of pure O2 breathing were present only when hyperoxia was initiated within the first 12 h, and the treatment was most efficacious when applied within the first 4 h (8). Moreover, even early administration of hyperoxia failed to exert major beneficial effects when the duration of the individual exposure was longer than 3 h (8). Unfortunately, this study (8) investigated an unresuscitated murine model of zymosan-induced shock, i.e., a condition of sterile systemic hyperinflammation, which certainly does not represent the clinical reality of septic shock. In this issue of Shock, Waisman et al. (9) now report on the effect of various dosing and timing-regimens of O2 breathing in rats with polymicrobial sepsis resulting from cecal ligation and puncture. The authors compared continuous 100% O2 breathing for 20 h, continuous 70% O2 breathing for 48 h, and intermittent 100% O2 breathing, the latter being applied for 6 h daily over 48 h. Videomicroscopy was used to assess pulmonary microvascular perfusion and capillary leakage, bronchoalveolar lavage fluid was obtained for measurement of cytokines and parameters of ROS and RNS release, and tissue morphometry allowed investigating lung inflammation and parenchymal condensation. Intermittent hyperoxia markedly attenuated lung injury and inflammation and did not aggravate oxidative or nitrosative stress. Of note, albeit hyperoxia was previously reported to cause macrocirculatory and microcirculatory vasoconstriction as a result of decreased local NO availability (10), it did not exert deleterious effects of capillary blood flow. In this context, the hyperoxia-induced respiratory acidosis may have assumed importance: Waisman et al. confirmed the well-known pure O2 breathing-induced fall in respiratory drive already described in conscious volunteers some decades ago, and this increase in arterial CO2 partial pressure (from 32 ± 3 mmHg in the air-breathing animals to 54 ± 6 mmHg in rats that had intermittent 100% O2) may have counterbalanced any hyperoxia-related microvascular constriction. Why can hyperoxia exert anti-inflammatory properties during systemic hyperinflammation after trauma and hemorrhage, sepsis, or ischemia/reperfusion injury? Clearly, at least during the acute phase, all these conditions are associated with tissue hypoxia resulting from circulatory depression and impaired microcirculatory perfusion. Furthermore, if present, additional blunt chest trauma causes (regional) alveolar hypoxia and, consecutively, hypoxemia. All these factors are known as most potent triggers of systemic inflammation (11), which could be counteracted by appropriately timed and dosed hyperoxia. The findings by Waisman et al. are fascinating, because they originate from a polymicrobial model of sepsis rather than a condition of sterile systemic hyperinflammation. Clearly, it can be argued that it has only limited clinical relevance because of the lacking resuscitative measures and the fact that the hyperoxic exposure was always initiated simultaneously with the induction of cecal ligation and puncture, i.e., nearly using a pretreatment design. Nevertheless, the authors have the merit of highlighting the potential benefit of pure O2 breathing during sepsis, provided the window of opportunity and the appropriate dosing are respected. Therefore, as far as (early and transitory) hyperoxia in sepsis is concerned: more than just enhanced O2 transport, it is time to recognize! ACKNOWLEDGMENTS Supported by the Deutsche Forschungsgemeinschaft (Klinische Forschergruppe 200 “Die Entzündungsantwort nach Muskulo-Skeletalem Trauma”).

CD4 + T-Helper Cells Stimulated in Response to Placental Ischemia Mediate Hypertension During Pregnancy

We have shown that hypertension in response to chronic placental ischemia is associated with elevated inflammatory cytokines and CD4(+) T cells. However, it is unknown whether these cells play an important role in mediating hypertension in response to placental ischemia. Therefore, we hypothesize that reduced uterine perfusion pressure (RUPP)-induced CD4(+) T cells increase blood pressure during pregnancy. To answer this question, CD4(+) T cells were isolated from spleens at day 19 of gestation from control normal pregnant (NP) and pregnant RUPP rats, cultured, and adjusted to 10(6) cells per 100 μL of saline for intraperitoneal injection into NP rats at day 13 of gestation. On day 18, in the experimental groups of rats, arterial catheters were inserted, and on day 19 mean arterial pressure was analyzed. Inflammatory cytokines and antiangiogenic factor soluble fms-like tyrosine kinase 1 were determined via ELISA. Mean arterial pressure increased from 104±2 mm Hg in NP rats to 124±2 mm Hg in RUPP rats (P<0.001) and to 118±1 mm Hg in rats receiving RUPP CD4(+) T cells (P<0.001). Circulating tumor necrosis factor-α and soluble fms-like tyrosine kinase 1 were elevated in recipients of RUPP CD4(+) T cells to levels similar to control RUPP rats. In contrast, virgin rats injected with NP or RUPP CD4(+) T cells exhibited no blood pressure changes compared with control virgin rats. Importantly, mean arterial pressure did not change in recipients of NP CD4(+) T cells (109±3 mm Hg). These data support the hypothesis that RUPP-induced CD4(+) T cells play an important role in the pathophysiology of hypertension in response to placental ischemia.

Mouse Hepatic Portal Venoconstrictive Response to Vasoconstrictors Is Much Weaker Than That in Rat

We previously reported that the portal venous pressure (PPV) response of perfused mouse livers to various vasoactive agents was much weaker than that of other mammals such as rat, rabbit, and guinea pigs. The purpose of this study was to determine the responsiveness of PPV in in vivo BALB/c mouse to intraportal injections of the 3 major vasoconstrictors of angiotensin II, norepinephrine, and endothelin-1 in comparison with that in Sprague-Dawley rats. In anesthetized spontaneously breathing animals, PPV, systemic arterial pressure, and central venous pressure were directly and continuously measured. The above-mentioned vasoconstrictors were injected into the portal vein as a bolus repetitively at the doses ranging 0.01-100 nmol/kg. A dose-dependent increase in systemic arterial pressure in response to each vasoconstrictor was observed similarly in both mice and rats. All vasoconstrictors also caused a dose-dependent increase in PPV in both species, but the peak levels in mouse did not reach higher than 7 mm Hg, whereas it reached as high as 15-24 mm Hg in rats. Immunostaining for alpha-smooth muscle actin revealed that smooth muscles were distributed substantially in portal venules of rat but scarcely in that of mouse. In conclusion, PPV response to various vasoconstrictors was limited in anesthetized BALB/c mice, as compared with the anesthetized Sprague-Dawley rats, presumably due to small amount of vascular smooth muscle in mouse portal venules.

Tissue pO 2 of Orthotopic 9L and C6 Gliomas and Tumor-Specific Response to Radiotherapy and Hyperoxygenation

Tumor hypoxia is a well-known therapeutic problem; however, a lack of methods for repeated measurements of glioma partial pressure of oxygen (pO(2)) limits the ability to optimize the therapeutic approaches. We report the effects of 9.3 Gy of radiation and carbogen inhalation on orthotopic 9L and C6 gliomas and on the contralateral brain pO(2) in rats using a new and potentially widely useful method, multisite in vivo electron paramagnetic resonance oximetry. Intracerebral 9L and C6 tumors were established in the left hemisphere of syngeneic rats, and electron paramagnetic resonance oximetry was successfully used for repeated tissue pO(2) measurements after 9.3 Gy of radiation and during carbogen breathing for 5 consecutive days. Intracerebral 9L gliomas had a pO(2) of 30-32 mm Hg and C6 gliomas were relatively hypoxic, with a pO(2) of 12-14 mm Hg (p < 0.05). The tissue pO(2) of the contralateral brain was 40-45 mm Hg in rats with either 9L or C6 gliomas. Irradiation resulted in a significant increase in pO(2) of the 9L gliomas only. A significant increase in the pO(2) of the 9L and C6 gliomas was observed in rats breathing carbogen, but this effect decreased during 5 days of repeated experiments in the 9L gliomas. These results highlight the tumor-specific effect of radiation (9.3.Gy) on tissue pO(2) and the different responses to carbogen inhalation. The ability of electron paramagnetic resonance oximetry to provide direct repeated measurements of tissue pO(2) could have a vital role in understanding the dynamics of hypoxia during therapy that could then be optimized by scheduling doses at times of improved tumor oxygenation.

The Role of Melatonin in the Pathogenesis of Hypertension in Rats With Metabolic Syndrome

Melatonin, the primary hormone of the pineal gland, is a known modulator of various physiological processes. The aim of this study was to evaluate the role of melatonin in the pathogenesis of hypertension in rats with metabolic syndrome and to assess whether melatonin supplementation prevents the development of hypertension in this model. Twenty male Sprague-Dawley (SD) rats were fed either a high fructose diet (n = 10) or a regular diet (control; n = 10) for 5 weeks. Urinary excretion of 6-hydroxymelatoninsulfate (a metabolite of melatonin) was measured at the beginning and the end of the study. An additional 20 SD rats were fed with the same diets but with a supplementation of melatonin (30 mg/kg/day) in their drinking water. Blood pressure (BP) was measured every week. BP increased significantly in rats fed with a high fructose diet and remained unchanged in the control group. The BP rise was associated with a significant decrease in melatonin secretion during sleep. Melatonin supplementation prevented the BP rise in fructose fed rats. BP increased by 14.6 +/- 1.0 mm Hg in the fructose fed rats, whereas it increased by only 3 +/- 2.6 mm Hg in rats fed with fructose and melatonin (P < 0.001 between groups). Melatonin secretion decreased in fructose fed rats that developed hypertension. Administration of melatonin blunted this BP rise. These data suggested that melatonin plays a role in the pathogenesis of hypertension in rats with metabolic syndrome.

Extracellular Renal Guanosine Cyclic 3′5′-Monophosphate Modulates Nitric Oxide– and Pressure-Induced Natriuresis

This study addresses the hypothesis that NO- and pressure-induced natriuresis are inhibited when guanosine cyclic 3',5'-monophosphate (cGMP) is prevented from being transported outside its renal synthesizing cells in vivo. Rats received a renal interstitial (RI) infusion of NO donor S-nitroso-N-acetylpenicillamine (SNAP) or SNAP+organic anion transporter inhibitor probenecid (PB) or SNAP+PB+cGMP. SNAP alone increased U(Na)V (P<0.05 at 1 hour and P<0.005 at 2 hours). In contrast, SNAP failed to increase U(Na)V when coinfused with PB, but cGMP coinfused with SNAP+probenecid restored the natriuretic response. SNAP alone increased RI cGMP (P<0.05) during the second experimental period. PB abolished the increase in RI cGMP in response to SNAP (P<0.01), but cGMP levels were restored by coinfusion with cGMP. PB also abolished SNAP-induced increases in fractional excretion of Na(+) (FE(Na)) and lithium (FE(Li)) (both P<0.01). PB also abolished the rise in RI cGMP and natriuresis induced by raising renal perfusion pressure (RPP) from 100 to 160 mm Hg in rats subjected to a standard pressure-natriuresis protocol and the natriuretic response was rescued by coinfusion with cGMP. RI administration of phosphodiesterase type V (PDE V) reduced both RIcGMP and U(Na)V in parallel (both P<0.01) without altering RIcAMP. The data demonstrate that export of cGMP from its renal synthesizing cells into the extracellular RI compartment is critical for the natriuretic action of NO donor SNAP or increased RPP and that RI cGMP controls basal Na(+) excretion. Extracellular cGMP modulates NO- and pressure-induced natriuresis.