Ali Reza Fathi

Safety and feasibility of long-term intravenous sodium nitrite infusion in healthy volunteers

Background Infusion of sodium nitrite could provide sustained therapeutic concentrations of nitric oxide (NO) for the treatment of a variety of vascular disorders. The study was developed to determine the safety and feasibility of prolonged sodium nitrite infusion. Methodology Healthy volunteers, aged 21 to 60 years old, were candidates for the study performed at the National Institutes of Health (NIH; protocol 05-N-0075) between July 2007 and August 2008. All subjects provided written consent to participate. Twelve subjects (5 males, 7 females; mean age, 38.8±9.2 years (range, 21–56 years)) were intravenously infused with increasing doses of sodium nitrite for 48 hours (starting dose at 4.2 µg/kg/hr; maximal dose of 533.8 µg/kg/hr). Clinical, physiologic and laboratory data before, during and after infusion were analyzed. Findings The maximal tolerated dose for intravenous infusion of sodium nitrite was 267 µg/kg/hr. Dose limiting toxicity occurred at 446 µg/kg/hr. Toxicity included a transient asymptomatic decrease of mean arterial blood pressure (more than 15 mmHg) and/or an asymptomatic increase of methemoglobin level above 5%. Nitrite, nitrate, S-nitrosothiols concentrations in plasma and whole blood increased in all subjects and returned to preinfusion baseline values within 12 hours after cessation of the infusion. The mean half-life of nitrite estimated at maximal tolerated dose was 45.3 minutes for plasma and 51.4 minutes for whole blood. Conclusion Sodium nitrite can be safely infused intravenously at defined concentrations for prolonged intervals. These results should be valuable for developing studies to investigate new NO treatment paradigms for a variety of clinical disorders, including cerebral vasospasm after subarachnoid hemorrhage, and ischemia of the heart, liver, kidney and brain, as well as organ transplants, blood-brain barrier modulation and pulmonary hypertension. Clinical Trial Registration Information http://www.clinicaltrials.gov; NCT00103025

Reversal of cerebral vasospasm via intravenous sodium nitrite after subarachnoid hemorrhage in primates

OBJECT Subarachnoid hemorrhage (SAH)-induced vasospasm is a significant underlying cause of aneurysm rupture-related morbidity and death. While long-term intravenous infusion of sodium nitrite (NaNO(2)) can prevent cerebral vasospasm after SAH, it is not known if the intravenous administration of this compound can reverse established SAH-induced vasospasm. To determine if the intravenous infusion of NaNO(2) can reverse established vasospasm, the authors infused primates with the compound after SAH-induced vasospasm was established. METHODS Subarachnoid hemorrhage-induced vasospasm was created in 14 cynomolgus macaques via subarachnoid implantation of a 5-ml blood clot. On Day 7 after clot implantation, animals were randomized to either control (saline infusion, 5 monkeys) or treatment groups (intravenous NaNO(2) infusion at 300 μg/kg/hr for 3 hours [7 monkeys] or 8 hours [2 monkeys]). Arteriographic vessel diameter was blindly analyzed to determine the degree of vasospasm before, during, and after treatment. Nitric oxide metabolites (nitrite, nitrate, and S-nitrosothiols) were measured in whole blood and CSF. RESULTS Moderate-to-severe vasospasm was present in all animals before treatment (control, 36.2% ± 8.8% [mean ± SD]; treatment, 45.5% ± 12.5%; p = 0.9). While saline infusion did not reduce vasospasm, NaNO(2) infusion significantly reduced the degree of vasospasm (26.9% ± 7.6%; p = 0.008). Reversal of the vasospasm lasted more than 2 hours after cessation of the infusion and could be maintained with a prolonged infusion. Nitrite (peak value, 3.7 ± 2.1 μmol/L), nitrate (18.2 ± 5.3 μmol/L), and S-nitrosothiols (33.4 ± 11.4 nmol/L) increased significantly in whole blood, and nitrite increased significantly in CSF. CONCLUSIONS These findings indicate that the intravenous infusion of NaNO(2) can reverse SAH-induced vasospasm in primates. Further, these findings indicate that a similar treatment paradigm could be useful in reversing cerebral vasospasm after aneurysmal SAH.

Carbon dioxide influence on nitric oxide production in endothelial cells and astrocytes: Cellular mechanisms

Cerebral vessels may regulate cerebral blood flow by responding to changes in carbon dioxide (CO(2)) through nitric oxide (NO) production. To better determine the role of NO production by human adult cerebral microvascular endothelial cells and human fetal astrocytes under different CO(2) conditions, we studied endothelial cell and astrocyte production of NO under hypo-, normo- and hypercapnic conditions. Human cerebral endothelial cell and fetal astrocyte cultures were exposed to hypocapnic (pCO(2) 21.7±6.7mmHg), normocapnic (pCO(2) 40.1±0.9mmHg) and hypercapnic (pCO(2) 56.3±8.7mmHg) conditions. NO production was recorded continuously over 24hours with stable pH. N-nitro-l-arginine [NLA; a nitric oxide synthase (NOS) inhibitor] and l-arginine (substrate for NO production via NOS) were used to further define the role of NOS in chemoregulation. NO levels in endothelial cells increased during hypercapnia by 36% in 8hours and remained 25% above baseline. NO increase in astrocytes was 30% after 1hour but returned to baseline at 8hours. NLA blocked NO increase in endothelial cells under hypercapnia. During hypocapnia, NO levels in the endothelial cells decreased by 30% at 8hours but were unchanged in astrocytes. l-arginine prevented NO decrease in endothelial cells under hypocapnia. NO changes in the endothelial cells correlated with changes in pCO(2) (R=0.99) and were independent of pH. This study suggests that cerebral endothelial cells and astrocytes release NO under normocapnic conditions and NO production is increased during hypercapnia and decreased during hypocapnia independent of pH. Further, this demonstrates that endothelial cells may play a pivotal role in chemoregulation by modulating NOS activity.

The role of nitric oxide donors in treating cerebral vasospasm after subarachnoid hemorrhage.

Reduced intra- and perivascular availability of nitric oxide (NO) significantly contributes to the multifactorial pathophysiology of cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH). The short half-life of NO demands its therapeutic substitution via NO donors. Classic NO donors such as sodium nitroprusside and nitroglycerin cannot be used as routine therapeutics because of serious side effects. Thus, a new generation of NO donors has been the subject of experimental investigations to avoid the drawbacks of the classic drugs. The purpose of this paper is to review the characteristics of different NO donors with regard to their promise and potential consequences in treating cerebral vasospasm. Additional novel concepts to increase NO concentrations, such as the activation of endothelial nitric oxide synthase (eNOS), are discussed.

The concept of a hybrid operating room: Applications in cerebrovascular surgery

The use of intraoperative digital substraction angiography (iDSA) is a tool in cerebrovascular surgery. According to recent studies, iDSA has been shown to alter surgical treatment in approximately 12% of cases. Moreover, it has been demonstrated that even experienced cerebrovascular surgeons might not accurately predict the need for iDSA. Intraoperative DSA prevents unnecessary surgical manipulations after occlusion of aneurysms and accurately demonstrates occlusion rates. We present our preliminary experience using routine iDSA within the concept of a hybrid operating room for cerebrovascular surgery. A total of 99 patients underwent iDSA in our hybrid operating room. Indications included intraoperative evaluation of occlusion rate of clipped aneurysms and patency of vicinity vessels (n = 82), chemical angioplasty with papaverin (n = 4), and balloon angioplasty (n = 1). In four (5%) patients, a reposition of the clip was needed due to neck remnant and perfusion of the aneurysm sack after clipping. A total of five cases underwent combined microsurgical and endovascular treatment of ruptured aneurysms and arteriovenous malformations (AVMs). The concept of a hybrid operating room has been considered in the planning and design of operation rooms dedicated to cerebrovascular surgery. Hybrid procedures combining endovascular with microsurgical strategies within the same surgical session are feasible and safe. These procedures are associated with cost-benefit advantages.

Estrogen Induces Nitric Oxide Production Via Nitric Oxide Synthase Activation in Endothelial Cells

INTRODUCTION 17β-estradiol (E2) has been found to induce vasodilation in the cardiovascular system and at physiological levels, resulting in prevention of cerebral vasospasm following subarachnoid hemorrhage (SAH) in animal models. The goal of this study was to analyze the cellular mechanism of nitric oxide (NO) production and its relation to E2, in vitro in brain and peripheral endothelial cells. METHODS Human umbilical endothelial cells (HUVEC) and brain endothelial cells (bEnd.3) were treated with estradiol (E2, 0.1, 10, 100, and 1,000 nM), and supernatant was collected at 0, 5, 15, 30, 60, and 120 min for nitric oxide metabolome (nitrite, NO₂) measurements. Cells were also treated with E2 in the presence of 1400W, a potent eNOS inhibitor, and ICI, an antagonist of estradiol receptors (ERs). Effects of E2 on eNOS protein expression were assessed with Western blot analysis. RESULTS E2 significantly increased NO2 levels irrespective of its concentration in both cell lines by 35 % and 42 % (p < 0.05). The addition of an E2 antagonist, ICI (10 μM), prevented the E2-induced increases in NO2 levels (11 % p > 0.05). The combination of E2 (10 nM) and a NOS inhibitor (1400W, 5 μM) inhibited NO2 increases in addition (4 %, p > 0.05). E2 induced increases in eNOS protein levels and phosphorylated eNOS (eNOS(p)). CONCLUSIONS This study indicates that E2 induces NO level increases in cerebral and peripheral endothelial cells in vitro via eNOS activation and through E2 receptor-mediated mechanisms. Further in vivo studies are warranted to evaluate the therapeutic value of estrogen for the treatment of SAH-induced vasospasm.

Topical application of 17β-estradiol (E2) improves skin flap survival through activation of endothelial nitric oxide synthase in rats

This study investigates the influence of 17β‐estradiol (E2) on nitric oxide (NO) production in endothelial cell cultures and the effect of topical E2 on the survival of skin flap transplants in a rat model. Human umbilical vein endothelial cells were treated with three different E2 concentrations and nitrite (NO 2) concentrations, as well as endothelial nitric oxide synthase (eNOS) protein expressions were analyzed. In vivo, random‐pattern skin flaps were raised in female Wistar rats 14 days following ovariectomy and treated with placebo ointment (group 1), E2 as gel (group 2), and E2 via plaster (group 3). Flap perfusion, survival, and NO 2 levels were measured on postoperative day 7. In vitro, E2 treatment increased NO 2 concentration in cell supernatant and eNOS expression in cell lysates (p < 0.05). In vivo, E2 treated (gel and plaster groups) demonstrated significantly increased skin flap survival compared to the placebo group (p < 0.05). E2 plaster‐treated animals exhibited higher NO 2 blood levels than placebo (p < 0.05) paralleling the in vitro observations. E2 increases NO production in endothelial cells via eNOS activation. Topical E2 application can significantly increase survival of ischemically challenged skin flaps in a rat model and may augment wound healing in other ischemic situations via activation of NO production.

Dimethyloxalylglycine stabilizes HIF-1α in cultured human endothelial cells and increases random-pattern skin flap survival in vivo.

Background: The goal of this study was to evaluate in vitro and in vivo the effects of up-regulation of the proangiogenic hypoxia inducible factor (HIF)-1&agr; induced by dimethyloxalylglycine on endothelial cell cultures and on skin flap survival. Methods: Human umbilical vein endothelial cell cultures were exposed to hypoxic conditions, to dimethyloxalylglycine, and to cobalt chloride for up to 24 hours. Expression of HIF-1&agr; and vascular endothelial growth factor (VEGF) in cell culture media was analyzed. In vivo, 20 male Wistar rats were assigned randomly to either the treatment group (dimethyloxalylglycine intraperitoneal injection, n = 10) or the control group (saline intraperitoneal injection, n = 10). A dorsal skin flap was raised in all animals and sutured back into place. Flap survival was evaluated on postoperative day 7 by laser Doppler and digital planimetry. Results: In vitro treatment of human umbilical vein endothelial cells during a 24-hour period showed a significant elevation of VEGF expression with dimethyloxalylglycine exposure (92 ± 35 pg/mg total cellular protein) or hypoxia exposure (88 ± 21 pg/mg total cellular protein) compared with controls (23 ± 10 pg/mg total cellular protein) (p < 0.05 for both). In vivo experiments showed a significant decrease of flap necrosis in the treatment group animals versus controls (35.95 ± 5.03 percent versus 44.42 ± 5.18 percent, p < 0.05). The laser Doppler evaluation revealed significantly increased blood flow in the proximal two-thirds of the flap in the treatment group compared with the control group (p < 0.05). Conclusion: Dimethyloxalylglycine treatment significantly increases VEGF and HIF-1&agr; expression in endothelial cell cultures and enhances skin flap survival in vivo in a rat model.

Patient specific “not” computer-assisted cranioplasty

Sir, We congratulate Dr. Bhargava et al. [1] for the development of another non-computerized technique for patient-specific cranioplasty and would like to add a few comments. This paper describes a well-designed technique to produce a titanium bone flap replica without the use of computer numerical controlled technique. This is especially of important value for delayed cranioplasty as it is applied for decompressive craniectomies [2, 3]. Additionally, titanium has certain advantages such as greater stability and diminished toxicity and weight compared to other grafts such as polymethyl-methacrylat, hydroxy-apatite, or polyester. However, one disadvantage of the presented technique and computer-assisted model techniques is that the replica may not be available during the same surgical procedure in case of open skull fractures, bone flap tumor invasion, calcified hematomas, etc [4, 5]. We have recently described a cost-effective patient-specific intraoperative molded cranioplasty using polymethyl-methacrylat [4]. This technique provides an immediate patient-specific, single-stage reconstruction, and avoids a second operation. We encourage Dr. Bhargava and colleagues to refine their technique in such a way that the titanium flap be made available during the initial surgical procedure in order to enhance the frequency and feasibility of this promising technique.

The Rabbit Blood Shunt Subarachnoid Haemorrhage Model

The recently introduced rabbit blood shunt subarachnoid haemorrhage model is based on the two standard procedures of subclavian artery cannulation and transcutaneous cisterna magna puncture. An extracorporeal shunt placed in between the arterial system and the subarachnoid space allows examiner-independent SAH in a closed cranium. Despite its straightforwardness, it is worth examining some specific features and characteristics of the model. We outline technical considerations to successfully perform the model with minimal mortality and morbidity. In addition, we discuss outcome measures, advantages and limitations, and the applicability of the model for the study of early brain injury and delayed cerebral vasospasm after SAH.