NMDA Receptor Activation Underlies the Loss of Spinal Dorsal Horn Neurons and the Transition to Persistent Pain after Peripheral Nerve Injury.

Peripheral nerve lesions provoke apoptosis in the dorsal horn of the spinal cord. The cause of cell death, the involvement of neurons, and the relevance for the processing of somatosensory information are controversial. Here, we demonstrate in a mouse model of sciatic nerve injury that glutamate-induced neurodegeneration and loss of γ-aminobutyric acid (GABA)ergic interneurons in the superficial dorsal horn promote the transition from acute to chronic neuropathic pain. Conditional deletion of Grin1, the essential subunit of N-methyl-d-aspartate-type glutamate receptors (NMDARs), protects dorsal horn neurons from excitotoxicity and preserves GABAergic inhibition. Mice deficient in functional NMDARs exhibit normal nociceptive responses and acute pain after nerve injury, but this initial increase in pain sensitivity is reversible. Eliminating NMDARs fully prevents persistent pain-like behavior. Reduced pain in mice lacking proapoptotic Bax confirmed the significance of neurodegeneration. We conclude that NMDAR-mediated neuron death contributes to the development of chronic neuropathic pain.

Contemporary Results of Aortic Coarctation Repair Through Left Thoracotomy.

BACKGROUND: Although surgical results for repair of coarctation of the aorta (CoA) have steadily improved, management of this condition remains controversial. The purposes of this study were to analyze the long-term outcomes of patients undergoing CoA repair through left thoracotomy and to define risk factors for reintervention. METHODS: All patients who were less than 18 years old and who underwent initial repair of CoA through left thoracotomy from 1995 to 2013 at Texas Children's Hospital (Houston, TX) were included. Patients were classified into 3 groups: 143 (42%) neonates (0 to 30 days old), 122 (36%) infants (31 days to 1 year old), and 78 (23%) older children (1 to 18 years old). Univariate and multivariate analyses were performed. RESULTS: A total of 343 patients (129 [38%] girls) with median age of 53 days (interquartile range [IQR],12 days to 9 months) and weight of 4.1 kg (IQR, 3.1 to 8.0) underwent repair with extended end-to-end anastomosis (291 patients [85%]), end-to-end anastomosis (44 patients [13%]), interposition graft (2 patients [0.6%]), or subclavian flap (6 patients [2%]). Concomitant diagnoses included genetic abnormalities (48 patients [14%]), isolated ventricular septal defects (58 patients [17%]), small left-sided structures (53 patients,16%), or other complex congenital heart disease (18 patients [5%]). Perioperative mortality was 1% (n = 4, all neonates). At a median follow-up of 6 years (7 days to 19 years), only 14 (4%) patients required reintervention (10 catheter-based procedures, 6 surgical repairs). A postoperative peak velocity of 2.5 m/s or greater was an independent risk factor for reintervention (odds ratio [OR], 4.0; 95% confidence interval [CI], 1.4 to 11.6). Within the cohort, 95 (33%) patients were hypertensive or remained on cardiac medications a median of 12 years (6 months to 19 years) after the surgical procedure. Development of perioperative hypertension was associated with higher risk of chronic hypertension or cardiac medication dependency (OR, 1.9; 95% CI, 1.1 to 3.3). CONCLUSIONS: CoA repair through left thoracotomy is associated with low rates of morbidity, mortality, and reintervention. Aortic arch obstruction should be completely relieved at the time of surgical intervention to minimize the risk of long-term recoarctation.

Risk stratification of patients with early breast cancer.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is the standard procedure performed to stage the axillae, and axillary node clearance (ANC) or radiotherapy is the treatment for nodal involvement. The aims of this study were to assess (1) the role of preoperative axillary ultrasonography (US), (2) the number of positive lymph nodes on ANC after either positive SLNB results or preoperative ultrasonographically guided nodal biopsy, and (3) the role of ANC in patients with node-positive breast cancer. PATIENTS AND METHODS: All patients with invasive breast cancer and axillary node involvement (but clinically negative nodes on presentation) who underwent ANC between January 2008 and December 2009 were identified, and information regarding clinicopathologic parameters and the nodal yield was collected. ANC was performed for 3 groups: patients with micrometastasis seen in SLNB specimens, macrometastasis seen in SLNB specimens, and positive axillary nodes detected on US biopsy. RESULTS: ANC was performed 141 times over the 2-year period. Forty-two percent of axillary node involvement was diagnosed by biopsy or preoperative US, and 40% of these patients received neoadjuvant chemotherapy. The remainder of cases were diagnosed by SLNB: 30% had micrometastases and 70% had macrometastases. Fifty percent of cancers with an ultrasonographic diagnosis of lymph node involvement were high grade and 56% had 4 or more positive nodes on ANC; this was significantly higher than in patients with positive SLNB results (P = .0001). Only 20% of patients with macrometastases on SLNB had 4 or more positive nodes in comparison with 56% with positive axillary lymph nodes by US (P < .0001). CONCLUSION: The routine use of preoperative axillary US and biopsy of abnormal nodes helps in identifying high-risk patients and thus aids in planning treatment.

Course, predictors of diaphragm recovery after phrenic nerve injury during pediatric cardiac surgery.

BACKGROUND: Hemidiaphragm paralysis from phrenic nerve injury is a known complication of congenital cardiac surgery. Return of diaphragm function has been reported; however, prior studies on this subject have been limited by small numbers, static assessment methods, or observation of plicated or non-plicated patients alone. To describe return of function, we reviewed fluoroscopy and ultrasonography in all diagnosed cases of diaphragmatic paralysis. METHODS: Surgical cases at our institution between 1991 and 2010 were identified for patients with postoperative hemidiaphragm paralysis diagnosed by chest X-ray, ultrasound, or fluoroscopy. Follow-up ultrasound and fluoroscopic studies were reviewed for return of diaphragm function. RESULTS: Seventy-two cases of postoperative hemidiaphragm paralysis were identified. Forty cases were plicated prior to discharge. Plicated patients were younger at time of diagnosis (median 46 days average 3.6 months; p=0.025) and had a larger proportion of single ventricle diagnoses (48% vs 16%) compared with non-plicated patients. Twenty-six patients with paralysis were excluded in follow-up due to lack of studies documenting diaphragm function after the diagnostic study. Of the remaining 46 cases, median follow-up was 353 days (range: 6 days to 17 years). Plicated and non-plicated patients regained function at similar frequency (60% and 54.8%, respectively). Plication status, Risk Adjustment for Congenital Heart Surgery (RACHS) 1 score, age at diagnosis, and side of paralysis did not predict failure of recovery. CONCLUSIONS: In the current era, return of diaphragm function after phrenic nerve injury sustained during congenital cardiac surgery is a known occurrence; however, predicting recovery continues to be difficult.

Is mode of presentation of B3 breast core biopsies (screen-detected or symptomatic) a distinguishing factor in the final histopathologic result or risk of diagnosis of malignancy?

BACKGROUND: The relation between histopathologic subclassification and mode of patient presentation (with a screen-detected vs. symptomatic lesion) with an abnormality in the breast core biopsy classified as having uncertain malignant potential (B3) has not been previously examined. We compared the histopathologic subclassification of these lesions and the frequency of malignancy in screen-detected and symptomatic patient groups. METHODS: All B3 core biopsies from one breast unit at the Royal Berkshire Hospital over a 5-year period (2006-2010) were analyzed (n = 131). After dividing the B3 biopsies into screen-detected and symptomatic groups, the National Health Service Breast Screening Programme histopathologic subclassification was used to further divide the groups into six subtypes. After surgery, a final diagnosis of invasive or in situ carcinoma was also noted. RESULTS: B3 classification comprised 3.8 % (131/3,440) of all core biopsies during that time period. There were 78 specimens from symptomatic (59 %) and 53 from screen-detected (41 %) patients. There was no statistically significant difference between papillary and fibroepithelial diagnoses between the two groups (47 vs. 42 %, p = 0.59, NS). There was no difference between the groups for atypia, lobular neoplasia, or sclerosing lesions (49 vs. 51 %, p = 0.8, NS). Cancer was found in 20 % of the symptomatic patients and in 17 % of the screen-detected group (p = 0.65, NS). CONCLUSIONS: Mode of patient presentation (with a screen-detected or symptomatic lesion) was not a distinguishing factor for breast histopathologic subclassification or for the final cancer diagnosis in patients whose breast core biopsy was classified as B3.

Occurrence and degree of intersex (testis-ova) in darters (Etheostoma SPP.) across an urban gradient in the Grand River, Ontario, Canada.

The variability and extent of the intersex condition (oocytes in testes, or testis-ova) was documented in fish along an urban gradient in the Grand River, Ontario, Canada, that included major wastewater treatment plant outfalls. A method for rapid enumeration of testis-ova was developed and applied that increased the capacity to quantify intersex prevalence and severity. Male rainbow darters (Etheostoma caeruleum) sampled downstream of the first major wastewater outfall (Waterloo) had a significant increase, relative to 4 upstream reference sites, in the mean proportion of fish with at least 1 testis-oocyte per lobe of testes (9-20% proportion with ≤ 1 testis-oocyte/lobe vs 32-53% and >1.4 testis-oocyte/lobe). A much higher mean incidence of intersex proportion and degree was observed immediately downstream of the second wastewater outfall (Kitchener; 73-100% and 8-70 testis-oocyte/lobe); but only 6.3 km downstream of the Kitchener outfall, the occurrence of intersex dropped to those of the reference sites. In contrast, downstream of a tertiary treated wastewater outfall on a small tributary, intersex was similar to reference sites. Estrogenicity, measured using a yeast estrogen screen, followed a similar pattern, increasing from 0.81 ± 0.02 ng/L 17b-estradiol equivalents (EEq) (Guelph), to 4.32 ± 0.07 ng/L (Waterloo), and 16.99 ± 0.40 ng/L (Kitchener). Female rainbow darter downstream of the Kitchener outfall showed significant decreases in gonadosomatic index and liver somatic index, and increases in condition factor (k) relative to corresponding reference sites. The prevalence of intersex and alterations in somatic indices suggest that exposure to municipal wastewater effluent discharges can impact endocrine function, energy use, and energy storage in wild fish.

Depth-profiling of environmental pharmaceuticals in biological tissue by solid-phase microextraction.

The parallel in vivo measurement of chemicals at various locations in living tissues is an important approach furthering our understanding of biological uptake, transportation, and transformation dynamics. However, from a technical perspective, such measurements are difficult to perform with traditional in vivo sampling techniques, especially in freely moving organisms such as fish. These technical challenges can be well addressed by the proposed depth-profiling solid-phase microextraction (DP-SPME) technique, which utilizes a single soft, flexible fiber with high spatial resolution. The analytical accuracy and depth-profiling capability of DP-SPME was established in vitro within a multilayer gel system and an onion artificially contaminated with pharmaceuticals. In vivo efficacy was demonstrated by monitoring pharmaceutical distribution and accumulation in fish muscle tissue. The DP-SPME method was validated against pre-equilibrium SPME (using multiple small fibers), equilibrium SPME, and liquid extraction methods; results indicated DP-SPME significantly improved precision and data quality due to decreased intersample variation. No significant adverse effects or increases in mortality were observed in comparisons of fish sampled by DP-SPME relative to comparable fish not sampled by this method. Consequently, the simplicity, effectiveness, and improved precision of the technique suggest the potential for widespread application of DP-SPME in the sampling of heterogeneous biotic and abiotic systems.

Continuous infusion of bupivacaine reduces postoperative morphine use in adolescent idiopathic scoliosis after posterior spine fusion.

STUDY DESIGN: Retrospective analysis. OBJECTIVE: To determine if an infusion of bupivacaine will reduce the need for intravenous opioids following posterior spine fusion. SUMMARY OF BACKGROUND DATA: Adolescent idiopathic scoliosis is estimated to occur with a frequency of 1% to 3% among the at-risk age group of 10 to 18 years. A small percentage of these patients will require surgical intervention. Data are limited regarding continuous infusion of local anesthetic after posterior spine fusion for pain control. METHODS.: Retrospective review of children 10 to 18 years with idiopathic scoliosis admitted to a tertiary care, 20-bed pediatric intensive care unit (P pediatric intensive care unit), following posterior spine fusion. The primary outcome was postoperative opioid use stratified by the presence of a catheter for continuous bupivacaine. Secondary outcomes included pain scores, side effect management, depth of catheter placement, and fluid resuscitation. RESULTS: Two hundred and forty-four children were eligible, 129 received a catheter for continuous bupivacaine, 115 did not. There were no differences in demographics. Significantly fewer patients receiving bupivacaine required a continuous basal infusion of morphine (32.6% vs. 85.2%, P < 0.001) resulting in an overall reduction opioid use on postoperative day 1 (18.9 vs. 26.4 mg, P < 0.001). Overall, pain scores were low in both groups. Limiting the analysis to only those with a bupivacaine catheter, the depth of catheter placement did not impact postoperative opioid use (P > 0.15). CONCLUSION: The use of a continuous infusion of bupivacaine provided good analgesia with low pain scores. The significant reduction in basal morphine use may reflect a replacement by bupivacaine, although this is limited by potential treatment bias. Multivariate analysis was required to control for ongoing changes in anesthesia practice over the many years of the study. The optimal depth of catheter placement is unclear from this analysis and should be studied prospectively.

Highly selective synthesis of oxabicycloalkanes by indium tribromide-mediated cyclization reactions of epoxyalkenes.

The cyclization of epoxyalkenes to oxabicycloalkanes is catalyzed by stoichiometric quantities of indium tribromide which exhibits excellent selectivity giving the oxabicyclic product in high yield in preference to other cyclized or rearrangement products.

Microplate orbital mixing improves high-throughput cell-based reporter assay readouts.

Reporter assays are commonly used for high-throughput cell-based screening of compounds, cDNAs, and siRNAs due to robust signal, ease of miniaturization, and simple detection and analysis. Among the most widely used reporter genes is the bioluminescent enzyme luciferase, which, when exposed to its substrate luciferin upon cell lysis, yields linear signal over a dynamic range of several orders of magnitude. Commercially available luciferase assay formulations have been developed permitting homogeneous, single-step cell lysis and reporter activity measurements. Assay conditions employed with these formulations are typically designed to minimize well-to-well luminescence variability due to variability in dispensing, evaporation, and incomplete sample mixing. The authors demonstrate that incorporating a microplate orbital mixing step into 96- and 384-well microplate cell-based luciferase reporter assays can greatly improve reporter readouts. They have found that orbital mixing using commercially available mixers facilitates maximal luciferase signal generation from high cell density-containing samples while minimizing variability due to partial cell lysis, thereby improving assay precision. The authors fully expect that widespread availability of mixers with sufficiently small orbits and higher speed settings will permit gains in signal and precision in the 1536-well format as well.

Persistence of bone marrow micrometastases in patients receiving adjuvant therapy for breast cancer: results at 4 years.

We have previously developed a quantitative PCR (QPCR) technique for the detection of cytokeratin 19 (CK19) transcripts in blood and bone marrow and compared this to immunocytochemistry (ICC). Together, both have shown promise for monitoring therapeutic efficacy in patients with metastatic breast cancer. The aim of this study was to determine the feasibility and value of these assays for minimal residual disease (MRD) in monitoring efficacy of adjuvant therapy following surgery for primary breast cancer. Bone marrow aspirates and peripheral blood samples were taken at the time of surgery from patients with primary breast cancer and no evidence of metastases on conventional scans. These were tested for the presence of CK19 mRNA transcripts and cytokeratin positive cells. Follow-up bone marrow aspirates were taken at 3, 6, 12, 24, 36 and 48 months. Prior to surgery, 51% of patients displayed evidence of disseminated cancer cells in the bone marrow by either or both QPCR and ICC. Of 91 patients who had repeat samples assayed, 87% and 65% had positive results at some time using QPCR and ICC, respectively. All patients received adjuvant systemic therapy and in 44 cases where there was a positive result in either the pretreatment or 3-month aspirate, 32/44 (73%) showed a fall in CK19:ABL ratio (QPCR) and 15/24 (63%) showed a reduction in the number of cytokeratin-positive cells (ICC) during follow-up. These results indicate that MRD persists despite adjuvant therapy in a majority of patients with primary breast cancer up to 4 years following surgery.

Genome-scale functional profiling of the mammalian AP-1 signaling pathway.

Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of approximately 20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin alpha1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis.