AMX - the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II.

The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 1012 photons s-1), micro-focus (7 µm × 5 µm), low-divergence (1 mrad × 0.35 mrad) energy-tunable (5-18 keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented.

Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II. Corrigendum.

A correction in the paper by Lazo et al. [(2021). J. Synchrotron Rad. 28, 1649-1661] is made.

Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II.

Here we present two robotic sample changers integrated into the experimental stations for the macromolecular crystallography (MX) beamlines AMX and FMX, and the biological small-angle scattering (bioSAXS) beamline LiX. They enable fully automated unattended data collection and remote access to the beamlines. The system designs incorporate high-throughput, versatility, high-capacity, resource sharing and robustness. All systems are centered around a six-axis industrial robotic arm coupled with a force torque sensor and in-house end effectors (grippers). They have the same software architecture and the facility standard EPICS-based BEAST alarm system. The MX system is compatible with SPINE bases and Unipucks. It comprises a liquid nitrogen dewar holding 384 samples (24 Unipucks) and a stay-cold gripper, and utilizes machine vision software to track the sample during operations and to calculate the final mount position on the goniometer. The bioSAXS system has an in-house engineered sample storage unit that can hold up to 360 samples (20 sample holders) which keeps samples at a user-set temperature (277 K to 300 K). The MX systems were deployed in early 2017 and the bioSAXS system in early 2019.

FMX - the Frontier Microfocusing Macromolecular Crystallography Beamline at the National Synchrotron Light Source II.

Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser. 425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser. 493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro-focusing Frontier MX beamline in sector 17-ID-2 at NSLS-II, covers a 5-30 keV photon energy range and delivers a flux of 4.0 × 1012 photons s-1 at 1 Šinto a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 1012 photons s-1 at final storage-ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods - serial crystallography on micrometre-sized crystals, raster optimization of diffraction from inhomogeneous crystals, high-resolution data collection from large-unit-cell crystals, room-temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample-screening and ligand-binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample-delivery methods have been implemented, such as an ultra-high-speed high-precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad. 25, 1362-1370], new microcrystal-optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238-246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun. 5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult-to-crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.

High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner.

The Frontier Microfocus Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II with its 1 µm beam size and photon flux of 3 × 1012 photons s-1 at a photon energy of 12.66 keV has reached unprecedented dose rates for a structural biology beamline. The high dose rate presents a great advantage for serial microcrystallography in cutting measurement time from hours to minutes. To provide the instrumentation basis for such measurements at the full flux of the FMX beamline, a high-speed, high-precision goniometer based on a unique XYZ piezo positioner has been designed and constructed. The piezo-based goniometer is able to achieve sub-100 nm raster-scanning precision at over 10 grid-linepairs s-1 frequency for fly scans of a 200 µm-wide raster. The performance of the scanner in both laboratory and serial crystallography measurements up to the maximum frame rate of 750 Hz of the Eiger 16M's 4M region-of-interest mode has been verified in this work. This unprecedented experimental speed significantly reduces serial-crystallography data collection time at synchrotrons, allowing utilization of the full brightness of the emerging synchrotron radiation facilities.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography.

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

Anterolateral Drawer Versus Anterior Drawer Test for Ankle Instability: A Biomechanical Model.

BACKGROUND: The addition of unconstrained internal rotation to the physical examination could allow for detection of more subtle degrees of ankle instability. We hypothesized that a simulated anterolateral drawer test allowing unconstrained internal rotation of the ankle would provoke greater displacement of the lateral talus in the mortise versus the anterior drawer test. METHODS: Ten cadaveric lower extremities were tested in a custom apparatus designed to reproduce the anterior drawer test and the anterolateral drawer test, in which the ankle was allowed to internally rotate about the intact deep deltoid ligament while being subluxed anteriorly. Specimens were tested intact and with anterior tibiofibular ligament sectioned. A differential variable reluctance transducer was used to measure lateral talar displacement with anterior forces of 25 and 50 N. RESULTS: No significant differences in talar displacement or ankle rotation were noted in intact specimens between the groups. Among sectioned specimens, significantly more talar displacement (25 N [6.5 ± 1.7 mm vs 3.8 ± 2.4 mm] and 50 N [8.7 ± 0.9 mm vs 4.5 ± 2.5 mm], P < .001) and ankle rotation (25 N [13.9 ± 8.0 degrees vs 0.0 ± 0.0 degrees] and 50 N [23.7 ± 5.8 degrees vs 0.0 ± 0.0 degrees], P < .001) were found in the anterolateral drawer versus anterior drawer group. CONCLUSION: In an ankle instability model, the anterolateral drawer test provoked almost twice the lateral talus displacement found with the anterior drawer test. CLINICAL RELEVANCE: Allowing internal rotation of the ankle while testing for ankle instability may allow the examiner to detect more subtle degrees of ankle instability.

Community-acquired Methicillin-resistant Staphylococcus aureus Musculoskeletal Infections: Emerging Trends Over the Past Decade.

BACKGROUND: The emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) has altered the management of pediatric musculoskeletal infections. Yet, institution-to-institution differences in MRSA virulence may exist, suggesting a need to carefully examine local epidemiological characteristics. The purpose of this study was to compare MRSA and methicillin-sensitive S. aureus (MSSA) musculoskeletal infections with respect to prevalence and complexity of clinical care over the past decade at a single children's hospital. METHODS: We retrospectively reviewed a series of patients presenting to The Children's Hospital of Philadelphia with a diagnosis of osteomyelitis, septic arthritis, or both over a 10-year period. Inclusion criteria were S. aureus (SA) infections proven by positive culture of blood, bone, or joint aspirate. Exclusion criteria were non-SA infectious etiologies. Hospital-acquired infections were also not included to exclusively evaluate acute, community-acquired cases. Data related to hospital course, laboratory values, and number of surgical interventions were collected and compared between MRSA and MSSA cohorts. RESULTS: In our series of pediatric patients, we identified 148 cases of acute, community-acquired musculoskeletal SA infections (MRSA, n=37 and MSSA, n=111). The prevalence of MRSA musculoskeletal infections increased from 11.8% in 2001 to 2002 to 34.8% in 2009 to 2010. Compared with MSSA, MRSA infections resulted in higher presenting C-reactive protein levels (10.4 vs. 7.8 mg/L, P=0.04), longer inpatient stays (10 vs. 5 d, P<0.01), multiple surgical procedures (n>1) (38% vs. 14%, P<0.01), increased sequelae (27% vs. 6%, P<0.01), and more frequent admissions to the intensive care unit (16% vs. 3%, P<0.01). CONCLUSIONS: At our institution over the past decade, we found an approximate 3-fold rise in community-acquired pediatric MRSA musculoskeletal infections accompanied by an elevated risk for complications during inpatient management. Awareness of the epidemiological trends of MRSA within the local community may guide parental counseling and facilitate timely and accurate clinical diagnosis and treatment. LEVEL OF EVIDENCE: Level II-prognostic retrospective study.

Macromolecular crystallography beamline X25 at the NSLS.

Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community.

Pharmacomechanical thrombolysis for phlegmasia cerulea dolens.

Phlegmasia cerulea dolens (PCD) is limb-threatening. Traditional treatments are very morbid. We examine the efficacy of percutaneous treatment of PCD. Between May 2005 and September 2008, we treated 21 limbs in 20 patients with lower extremity PCD who were candidates for thrombolysis. Diagnosis was by clinical examination and duplex ultrasound. Catheter access to the deep venous system was obtained through a popliteal vein. Therapy used pulse spray thrombolysis with tissue plasminogen activator (tPA). Infusion catheters and adjunctive percutaneous techniques were used as indicated. Postoperatively, patients were treated with systemic anticoagulation, compression hose, and interval follow-up. Limbs were graded according to the CEAP classification. Twenty patients (13 male) were treated with a mean age of 55.8 years. Nine patients had hypercoagulable states, four May Thurner syndrome, three a history of cancer, one postcolon resection, one acute myocardial infarction, and one postfemoral vein puncture. All patients had resolution of PCD without the need for open surgery. The initial tPA dose was 19.5 mg with pulse spray thrombolysis. Infusion catheters were required in 18 patients and used for 16.1 hours (range, 8 to 36 hours) until complete thrombolysis. Venous angioplasty was necessary in 14 patients with nine of these requiring venous stents. One patient required above-knee amputation despite successful treatment of her PCD. Mean follow-up was 10.7 months (range, 1 to 39 months). All patients demonstrated no or minimal residual thrombus and intact valvular function and a mean clinical CEAP score of 2.4. Percutaneous treatment of PCD produced excellent results with minimal morbidity.

Balloon-expandable covered stent therapy of complex endovascular pathology.

The current study was designed to investigate our hypotheses that balloon-expandable covered stents display acceptable function over longitudinal follow-up in patients with complex vascular pathology and provide a suitable alternative for the treatment of recurrent in-stent restenosis. All stents were Atrium iCast, which is a balloon-mounted, polytetrafluoroethylene-covered stent with a 6F/7F delivery system. A retrospective review was performed of 49 patients with 66 stented lesions. Data were analyzed with life tables and t-tests. The most commonly treated vessels were the iliac (61%) and renal (24%) arteries. Indications for covered stent placement were unstable atheromatous lesions (50%), recurrent in-stent restenosis (24%), aneurysm (8%), aortic bifurcation reconstruction (7.5%), dissection (4.5%), endovascular aneurysm repair-related (4.5%), and stent fracture (1.5%). Patency was assessed by angiogram or duplex ultrasonography. The primary end point was patency and secondary end points were technical success and access-site complications. Mean follow-up was 13 months (range 1.5-25). The technical success rate was 97%. Unsuccessful outcomes were due to deployment error (n=1) and stent malpositioning (n=1). The cohort (n=64) 6- and 12-month primary patency rates were 96% and 84%, respectively. Twelve-month assisted primary patency was 98%. Iliac artery stents (n=38) had a primary patency of 97% at 6 months and 84% at 12 months with an assisted primary patency of 100% at 12 months. Renal artery stents (n=16) had a primary patency of 92% at 6 months and 72% at 12 months with an assisted primary patency of 92% at 6 and 12 months. Stents placed for recurrent in-stent restenosis (n=16) had a primary patency of 85%, assisted primary patency of 93%, and a 15% restenosis rate at 12 months. Specifically, stents placed for renal artery recurrent in-stent restenosis (n=10) had a primary patency of 73%, assisted primary patency of 82%, and a restenosis rate of 27%. The restenosis rate included two renal artery occlusions in patients noncompliant with clopidogrel use and resulted in ipsilateral kidney loss in both patients. In-stent peak systolic velocities decreased significantly (p<0.05) from preoperation to 12 months in iliac stents and to 18 months in renal stents. Ankle-brachial index increased significantly in iliac stents from preoperation (0.62+/-0.18) to 18 months (0.86+/-0.16). Successful exclusion of atheromatous lesions and aneurysm/dissection/endoleak was 100%. Access-site complications occurred in 6%: pseudoaneurysm (n=2), dissection (n=1), and bleeding (n=1). Balloon-expandable covered stents have an acceptable primary patency with an excellent assisted patency after salvage angioplasty. The clinical utility of this technology is broad for the treatment of aneurysms, extravasation, unstable atheromatous lesions, and recurrent in-stent restenosis.

Saphenous vein graft aneurysm with graft-enteric fistula after renal artery bypass.

A 65-year-old female presented with upper gastrointestinal hemorrhage thirty years following an aorta-to-right renal artery bypass constructed with saphenous vein. Upper endoscopy demonstrated a duodenal ulcer, and a CAT scan demonstrated aneurysmal degeneration of her renal artery bypass with duodenal impingement. Laparotomy demonstrated erosion of the aneurysm through the posterior wall of the duodenum; extra-anatomic renovascular reconstruction and primary duodenal repair was performed. Although aneurysmal degeneration of intraabdominal saphenous vein grafts is well described and rupture likewise reported, this report represents the first description of an intraabdominal autogenous vein graft aneurysm presenting with gastrointestinal erosion and fistula.

External fixation of high-energy tibia fractures.

External fixation (EF) of tibia fractures has been associated with nonunions and malunions at our large pediatric trauma center. This study was designed to determine the successes and shortcomings of EF, especially with respect to maintenance of alignment and time to union. We believe that this will contribute to the limited amount of literature examining the complications associated with this treatment modality in the pediatric population. Thirty-one consecutive high-energy tibia fractures treated with EF over 4.5 years were analyzed. There were 22 boys and 9 girls (4-17 years old; mean, 11.9 years). Mean length of follow-up was 15 months. Of the 31 fractures analyzed, 19 were open fractures (12 closed, 3 grade I, 9 grade II, and 7 grade III). Of 30 fractures, 3 required skin graft, whereas 7 required fasciotomy. Mean duration of EF was 3.2 months. Mean time to union was 4.8 months. For complication rates, 4 of 30 had delayed union, 2 of 30 had nonunion, 8 of 30 had minor malunion, 3 of 30 had major malunion, 3 of 30 had leg length discrepancy, 8 of 30 had pin track infection, 3 of 30 had wound infection, 2 of 30 had osteomyelitis, and 4 of 30 required surgery for nonunion. Time to union differed between those aged 11 years or younger and those aged 12 years or older (means of 3.2 and 6.0 months, respectively; P = 0.001). Union time also differed between those with closed or grade I open fractures and those with grade II or III open fractures (3.9 and 5.7 months, respectively; P = 0.035). Leg length discrepancy rate differed between children aged 11 years or younger and those aged 12 years or older (3/13 and 0/18, respectively; P = 0.05). Although EF has been touted as the standard treatment of high-energy pediatric tibia fractures, our close analysis revealed a high rate of problems such as long union times (especially ages >or=12), malunion, leg length discrepancy (especially ages

Loss of renal function and microvascular blood flow after suprarenal aortic clamping and reperfusion (SPACR) above the superior mesenteric artery is greatly augmented compared with SPACR above the renal arteries.

OBJECTIVE: Renal insufficiency continues to be a complication that can affect patients after treatment for suprarenal aneurysms and renal artery occlusive disease. To our knowledge, no data are available showing that suprarenal aortic clamping and reperfusion (SRACR) above the renal arteries (renal-SRACR) preserves renal function compared with SRACR above the superior mesenteric artery (SMA-SRACR). This study examined the hypothesis that SMA-SRACR-induced downregulation of renal blood flow and function is more severe than renal-SRACR owing to the addition of systemic oxygen-derived free radical (ODFR) release. METHODS: Male Sprague-Dawley rats (about 350 g) were anesthetized and microdialysis probes or laser Doppler fibers were inserted into the renal cortex (depth of 2 mm) and into the renal medulla (depth of 4 mm). Laser Doppler blood flow was continuously monitored, and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 microL/min with lactated Ringer's solution. RESULTS: SMA-SRACR and Renal-SRACR decreased medullary and cortical blood flow and nitric oxide (NO) synthesis. SMA-SRACR downregulated cortical inducible NO synthase, whereas renal-SRACR did not. The cortex and medulla responded to the decreased blood flow and NO synthesis by increasing in prostaglandin E2 synthesis, which was due to increased cyclooxygenase-2 content. Superoxide dismutase restored SMA-SRACR (but not renal-SRACR) cortical and medullary NO synthesis, suggesting that ODFRs generated during mesenteric ischemia-reperfusion were one of the systemic mechanisms contributing to decreased renal NO synthesis in the SMA-SRACR model. The 90% decrease in creatinine clearance after SMA-SRACR was greater than the 60% decrease after renal-SRACR. CONCLUSIONS: These data show that NO is important in maintaining renal cortical and medullary blood flow and NO synthesis after renal and SMA-SRACR. These data also suggest that in addition to the renal ischemia-reperfusion caused by both models, SMA SRACR induces mesenteric ischemia-reperfusion, resulting in the generation of ODFRs, which contribute to decreased renal cortical and medullary NO synthesis. Maintaining splanchnic blood flow or attempting to keep SRACR below the SMA level may be helpful in developing strategies to minimize the renal injury after SRACR.

Preliminary results of subintimal angioplasty for limb salvage in lower extremities with severe chronic ischemia and limb-threatening ischemia.

OBJECTIVE: This study examined the hypothesis that superficial femoral artery (SFA) subintimal angioplasty (SI-PTA) can maintain limb salvage with minimal complications in patients with symptomatic occlusive arterial disease. METHODS: From March 1, 2004, until April 28, 2006, 78 patients with rest pain (62.2%), gangrene (25.6%), or severe progressive claudication (12.2%) were treated consecutively with 82 SFA SI-PTAs (4 bilateral). The mean age was 59 +/- 1.2 years, and 21 (27%) of the patients were female. All patients were treated in the operating room under local anesthesia by using fluoroscopic guidance, and the percentage SFA that was occluded was measured during the diagnostic portion of the procedure. Selective stent placement was performed after successful recanalization of the occluded arterial segments. Patients were treated with chronic aspirin and clopidogrel bisulfate for 3 months and followed up at 30 days and then every 3 months with physical examination and arterial duplex scan. RESULTS: Of the 82 SFA SI-PTA attempts, 76 (92%) were initially successful, with an increase in the ankle-brachial index from 0.46 +/- 0.02 to 0.88 +/- 0.01 (P < .001). Five of the six patients with a failed SFA SI-PTA were female, two of the six had had previous bypass attempts, and one of the six had had a previous SFA SI-PTA attempt by another physician. Forty-nine (64%) of the 76 initially successful SFA SI-PTAs required placement of a stent, and 43 (56.5%) of the successful 76 SFA SI-PTAs required additional PTA of 1 or more arterial segments. The group treated with a successful SFA SI-PTA had 42.5% +/- 3.5% SFA occlusion, compared with 82% +/- 10% (P < .05) in the group with a failed attempt at SFA SI-PTA. Two of the six patients with initial SI-PTA failure underwent leg amputation within 30 days, three were treated with successful leg bypass surgery, and one was lost to follow-up. Of the 76 successful SFA SI-PTAs, 5 (6.5%) failed within 90 days, and the patients were treated successfully with leg bypass surgery. Of the 71 limbs with patent SI-PTAs at 90 days, 68 have remained patent with a mean follow-up 10.4 +/- 0.7 months (range, 2-24 months). Three of the 71 SFA SI-PTAs failed between 4 and 7 months (mean, 5 +/- 0.7 months): 1 patient was treated with successful bypass surgery, 1 patient is currently considering further intervention, and 1 patient was treated with amputation. Ten (14%) of the 71 successful SFA SI-PTAs required limited PTA for asymptomatic restenosis, as identified by the arterial duplex scan (7.4 +/- 1.4 months; range, 2-16 months). There were no perioperative deaths, and three patients have died during follow-up with patent SFA SI-PTAs (9.3 +/- 1.4 months). CONCLUSIONS: These data suggest that SFA SI-PTA can be successfully used for limb salvage with minimal morbidity and mortality in a group of patients with severe lower extremity occlusive vascular disease.

Iodinated contrast induced renal vasoconstriction is due in part to the downregulation of renal cortical and medullary nitric oxide synthesis.

OBJECTIVES: The loss of renal function continues to be a frequent complication of the iodinated contrast agents used to perform diagnostic angiography and endovascular procedures. This study examined the hypothesis that contrast-induced renal injury is partly due to a decrease in cortical and medullary microvascular blood flow after the downregulation of endogenous renal cortical and medullary nitric oxide (NO) synthesis. METHODS: Anesthetized male Sprague-Dawley rats (300 g) had microdialysis probes or laser Doppler fibers inserted into the renal cortex to a depth of 2 mm and into the renal medulla to a depth of 4 mm. Laser Doppler blood flow was continuously monitored, and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 muL/min with lactated Ringer's solution. Dialysate fluid was collected at time zero (basal) and 60 minutes after infusion of either saline or Conray 400 (6 mL/kg). Both groups were treated with saline carrier, N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 30 mg/kg), L-arginine (400 mg/kg), or superoxide dismutase (10,000 U/kg), an oxygen-derived free radical scavenger. Dialysate was analyzed for total NO and eicosanoid synthesis. The renal cortex and medulla were analyzed for inducible NO synthase (iNOS), cyclooxygenase-2 (COX2), prostacyclin synthase, and prostaglandin E(2) (PGE(2)) synthase content by Western blot analysis. RESULTS: Conray caused a marked decrease in cortical and medullary blood flow with a concomitant decrease in endogenous cortical NO, PGE(2), and medullary NO synthesis. The addition of L-NAME to the Conray further decreased cortical and medullary blood flow and NO synthesis, which were restored toward control by L-arginine. Neither L-NAME nor L-arginine (added to the Conray) altered cortical or medullary eicosanoids release. Medullary PGE(2) synthesis decreased when superoxide dismutase was added to the Conray treatment, suggesting that oxygen-derived free radicals had a protective role in maintaining endogenous medullary PGE(2) synthesis after Conray treatment. Conray did not significantly alter iNOS, COX-2, prostacyclin synthase, or PGE(2) synthase content. CONCLUSIONS: These findings suggest that the downregulation of renal cortical and medullary NO synthesis contributes to the contrast-induced loss of renal cortical and medullary microvascular blood flow. Preservation of normal levels of renal cortical and medullary NO synthesis may help prevent or lessen contrast-induced renal vasoconstriction and lessen contrast-induced renal injury found after diagnostic and therapeutic endovascular procedures.

Oxygen-radical regulation of renal blood flow following suprarenal aortic clamping.

OBJECTIVE: Renal insufficiency continues to be complication that can affect patients after treatment for suprarenal aneurysms and renal artery occlusive disease. One proposed mechanism of renal injury after suprarenal aortic clamping (above the superior mesenteric artery) and reperfusion (SMA-SRACR) is the loss of microvascular renal blood flow with subsequent loss of renal function. This study examines the hypothesis that the loss of medullary and cortical microvascular blood flow following SMA-SRACR is due to oxygen-derived free radical down-regulation of endogenous medullary and cortical nitric oxide synthesis. METHODS: Anesthetized male Sprague-Dawley rats (about 350 g) either had microdialysis probes or laser Doppler fibers inserted into the renal cortex (depth of 2 mm) and into the renal medulla (depth of 4 mm). Laser Doppler blood flow was continuously monitored. The microdialysis probes were connected to a syringe pump and perfused in vivo at 3 microL/min with lactated Ringer's solution. The animals were subjected to SMA-SRACR (or sham) for 30 minutes, followed by 60 minutes of reperfusion. Laser Doppler blood flow after the 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion was compared with the time zero (basal) and with the corresponding sham group and reported as percent change compared with the time zero baseline. The microdialysis fluid was collected at time zero (basal) and compared with the dialysis fluid collected after 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion as well as the corresponding sham group. The microdialysis dialysate was analyzed for total nitric oxide (microM) and prostaglandin E2 (PGE2), 6-keto-PGF(1alpha) (PGI2 metabolite), and thromboxane B2 synthesis. The data are reported as percent change compared with the baseline time zero. The laser Doppler blood flow and microdialysis groups were treated with either saline carrier, N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME) (30 mg/kg, nitric oxide synthesis inhibitor), L-arginine (400 mg/kg, nitric oxide precursor), superoxide dismutase (SOD, 10,000 U/kg, oxygen-derived free radical scavenger), L-NAME + SOD, or L-arginine + SOD. SOD was given 30 minutes before the reperfusion, and the other drugs were given 15 minutes before reperfusion. The renal cortex and medulla were separated and analyzed for inducible nitric oxide synthase (iNOS), cyclooxygenase-2, prostacyclin synthase, and PGE2 synthase content by Western blot. RESULTS: Superior mesenteric artery-SRACR caused a marked decrease in medullary and cortical blood flow with a concomitant decrease in endogenous medullary and cortical nitric oxide synthesis. These changes were further accentuated by L-NAME treatment but restored toward sham levels by L-arginine treatment after SMA-SRACR. The kidney appeared to compensate for these changes by increasing cortical and medullary PGE2 synthesis and release. SOD treatment restored renal cortical and medullary nitric oxide synthesis and blood flow in the ischemia-reperfusion group and in the ischemia-reperfusion group treated with L-NAME. CONCLUSIONS: These data show that nitric oxide is important in maintaining renal cortical and medullary blood flow and nitric oxide synthesis. These data also support the hypothesis that the loss of medullary and cortical microvascular blood flow following SRACR is due in part to oxygen-derived free radical downregulation of endogenous medullary and cortical nitric oxide synthesis.