Methods for implantation of micro-wire bundles and optimization of single/multi-unit recordings from human mesial temporal lobe.

OBJECTIVE: The authors report methods developed for the implantation of micro-wire bundles into mesial temporal lobe structures and subsequent single neuron recording in epileptic patients undergoing in-patient diagnostic monitoring. This is done with the intention of lowering the perceived barriers to routine single neuron recording from deep brain structures in the clinical setting. APPROACH: Over a 15 month period, 11 patients were implanted with platinum micro-wire bundles into mesial temporal structures. Protocols were developed for (A) monitoring electrode integrity through impedance testing, (B) ensuring continuous 24-7 recording, (C) localizing micro-wire position and 'splay' pattern and (D) monitoring grounding and referencing to maintain the quality of recordings. MAIN RESULTS: Five common modes of failure were identified: (1) broken micro-wires from acute tensile force, (2) broken micro-wires from cyclic fatigue at stress points, (3) poor in vivo micro-electrode separation, (4) motion artifact and (5) deteriorating ground connection and subsequent drop in common mode noise rejection. Single neurons have been observed up to 14 days post-implantation and on 40% of micro-wires. SIGNIFICANCE: Long-term success requires detailed review of each implant by both the clinical and research teams to identify failure modes, and appropriate refinement of techniques while moving forward. This approach leads to reliable unit recordings without prolonging operative times, which will help increase the availability and clinical viability of human single neuron data.

Effect of insurance status on postacute care among working age stroke survivors.

OBJECTIVE: Utilization of postacute care is associated with improved poststroke outcomes. However, more than 20% of American adults under age 65 are uninsured. We sought to determine whether insurance status is associated with utilization and intensity of institutional postacute care among working age stroke survivors. METHODS: A retrospective cross-sectional study of ischemic stroke survivors under age 65 from the 2004-2006 Nationwide Inpatient Sample was conducted. Hierarchical logistic regression models controlling for patient and hospital-level factors were used. The primary outcome was utilization of any institutional postacute care (inpatient rehabilitation or skilled nursing facilities) following hospital admission for ischemic stroke. Intensity of rehabilitation was explored by comparing utilization of inpatient rehabilitation facilities and skilled nursing facilities. RESULTS: Of the 33,917 working age stroke survivors, 19.3% were uninsured, 19.8% were Medicaid enrollees, and 22.8% were discharged to institutional postacute care. Compared to those privately insured, uninsured stroke survivors were less likely (adjusted odds ratio [AOR] 0.53, 95% confidence interval [CI] 0.47-0.59) while stroke survivors with Medicaid were more likely to utilize any institutional postacute care (AOR = 1.40, 95% CI 1.27-1.54). Among stroke survivors who utilized institutional postacute care, uninsured (AOR = 0.48, 95% CI 0.36-0.64) and Medicaid stroke survivors (AOR = 0.27, 95% CI 0.23-0.33) were less likely to utilize an inpatient rehabilitation facility than a skilled nursing facility compared to privately insured stroke survivors. CONCLUSIONS: Insurance status among working age acute stroke survivors is independently associated with utilization and intensity of institutional postacute care. This may explain differences in poststroke outcomes among uninsured and Medicaid stroke survivors compared to the privately insured.

Enrollment of women and minorities in NINDS trials.

OBJECTIVE: To determine policy-associated changes over time in 1) the enrollment of women and minorities in National Institute of Neurological Disorders and Stroke (NINDS)-funded clinical trials and 2) the trial publication reporting of race/ethnicity and gender. METHODS: All NINDS-funded phase III trials published between 1985 and 2008 were identified. Percent of African Americans, Hispanic Americans, and women enrolled in the trials was calculated for those trials with available data. Z tests were used to compare reporting and enrollment data from before (period 1) and after (period 2) 1995 when NIH enacted their policies regarding race, ethnicity, and gender. Percent of main trial publications reporting enrollment of African Americans, Hispanic Americans, and women was also calculated. RESULTS: Of the 56 trials identified, 100%, 48%, and 25% reported enrollment by gender, race, and ethnicity. Women constituted 42.1% of the trial population. Enrollment of women increased over time (36.9% period 1; 49.0% period 2, p < 0.001). African Americans constituted 19.8% of the enrollees in trials with available data and enrollment increased over time (11.6% period 1; 30.7% period 2, p < 0.001). Hispanic Americans constituted 5.8% of subjects in trials with available data and enrollment decreased over time (7.4% period 1; 5.0% period 2, p < 0.001). CONCLUSIONS: Improvements in reporting of race/ethnicity in publications and enrollment of Hispanics in NINDS trials are needed. While African American representation is above population levels, Hispanic Americans are underrepresented in NINDS trials and representation is declining despite Hispanics' increasing representation in the US population.

Critical analysis of a computer-assisted tutorial on ECG interpretation and its ability to determine competency.

BACKGROUND: We developed a computer-based tutorial and a posttest on ECG interpretation for training residents and determining competency. METHODS: Forty residents, 6 cardiology fellows, and 4 experienced physicians participated. The tutorial emphasized recognition and understanding of abnormal ECG features. Active learning was promoted by asking questions prior to the discussion of ECGs. Interactivity was facilitated by providing rapid and in-depth rationale for correct answers. Responses to questions were recorded and extensively analyzed to determine the quality of questions, baseline knowledge at different levels of training and improvement of grades in posttest. Posttest grades were used to assess improvement and to determine competency. RESULTS: The questions were found to be challenging, fair, appropriate and discriminative. This was important since the quality of Socratic questions is critical for the success of interactive programs. The information on strengths and weakness in baseline knowledge at different levels of training were used to adapt our training program to the needs of residents. The posttest revealed that the tutorial contributed to marked improvement in feature recognition. Competency testing distinguished between residents with outstanding grades and those who needed remediation. CONCLUSIONS: The strategy for critical evaluation of our computer program could be applied to any computer-based educational program, regardless of topic.

Identification of low-abundance differentially expressed transcripts using arrayed cDNA clones.

Analysis of comparative gene expression by the use of DNA microarrays has become a widely used tool. However, this technique is only readily applied to organisms where sequence information is known. This paper describes the development of a low-cost method of gene discovery by enrichment of differentially expressed transcripts, which uses cDNA library arrays of bacterial clones on nylon membranes (macroarrays) coupled with a subtractive probe preparation method to discover differentially expressed genes. The method requires no prior knowledge of the organism's genome sequence and overcomes the inherent insensitivity of standard methods of macroarray hybridisation.

Regulation of the pharynx of Caenorhabditis elegans by 5-HT, octopamine, and FMRFamide-like neuropeptides.

More than fifty FMRFamide-like neuropeptides have been identified in nematodes. We addressed the role of a subset of these in the control of nematode feeding by electrophysiological recording of the activity of C. elegans pharynx. AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), AF8 (KSAYMRFamide), and GAKFIRFamide (encoded by the C. elegans genes flp-8, flp-14, flp-6, and flp-5, respectively) increased pharyngeal action potential frequency, in a manner similar to 5-HT. In contrast, SDPNFLRFamide, SADPNFLRFamide, SAEPFGTMRFamide, KPSVRFamide, APEASPFIRFamide, and AQTVRFamide (encoded by the C. elegans genes flp-1; flp-1; flp-3; flp-9; flp-13, and flp-16, respectively) inhibited the pharynx in a manner similar to octopamine. Only three of the neuropeptides had potent effects at low nanomolar concentrations, consistent with a physiological role in pharyngeal regulation. Therefore, we assessed whether these three peptides mediated their actions either directly on the pharynx or indirectly via the neural circuit controlling its activity by comparing actions between wild-type and mutants with deficits in synaptic signaling. Our data support the conclusion that AF1 and SAEPFGTMRFamide regulate the activity of the pharynx indirectly, whereas APEASPFIRFamide exerts its action directly. These results are in agreement with the expression pattern for the genes encoding the neuropeptides (Kim and Li, 1999) as both flp-8 and flp-3 are expressed in extrapharyngeal neurons, whereas flp-13 is expressed in I5, a neuron with synaptic output to the pharyngeal muscle. These results provide the first, direct, functional information on the action of neuropeptides in C. elegans. Furthermore, we provide evidence for a putative inhibitory peptidergic synapse, which is likely to have a role in the control of feeding.

The initial attitudes of patients toward longer maintenance hemodialysis.

UNLABELLED: Longer hemodialysis (HD) as practiced in parts of Europe and Japan may improve both blood pressure control and patient survival. Nevertheless, in the USA, the trend has been to shorten dialysis time using larger dialyzers and increased blood flows. Many patients find the notion of shorter dialysis enticing. Most are unaware ofthe potential benefits of longer dialysis. We surveyed stable chronic HD patients in an urban area, the vast majority of whom received conventional 4-hour treatments, regarding their attitude toward extending their dialysis time to 5 hours. They were informed that longer dialysis has been associated with better blood pressure control and improved survival. One hundred and sixteen patients completed questionnaires during a single dialysis session. Forty-six (40%) agreed to extended dialysis while 70 (60%) did not. There was no difference between the groups with respect to the following variables: age, race, etiology of ESRD, time on dialysis, marital status, number of children at home, number residing in the household, education, or employment status. Male gender was associated with a positive response (p = 0.03). Various suggested and spontaneous reasons were given for a negative response. CONCLUSION: With minimally detailed information, 4 in 10 patients were willing to extend their treatment time to 5 hours in the hope of improving morbidity and survival. No sociodemographic variable except gender was associated with a positive response.

Neuronal expression of an FMRFamide-gated Na+ channel and its modulation by acid pH.

The molluscan Phe-Met-Arg-Phe-amide (FMRFamide)-gated sodium channels (FaNaCs) show both structural and functional similarities to the mammalian acid-sensing ion channels (ASICs). Both channel types are related to the epithelial sodium channels and, although the neuropeptide FMRFamide directly gates the FaNaCs, it also modulates the proton-gating properties of ASICs. It is not yet known whether protons can alter the gating properties of the FaNaCs. We chose to examine this possibility at a site of FaNaC expression in the nervous system of the mollusk Lymnaea stagnalis. We cloned a putative L. stagnalis FaNaC (LsFaNaC) that exhibited a high degree of sequence identity to the Helix aspersa FaNaC (HaFaNaC, 60%), and a weaker homology to the ASICs (ASIC3, 22%). In situ hybridization was used to map the LsFaNaC expression pattern in the brain and to identify the right pedal giant1 (RPeD1) neuron as a site where the properties of the endogenous channel could be studied. In RPeD1 neurons isolated in culture, we demonstrated the presence of an FMRFamide-gated sodium current with features expected for a FaNaC: amiloride sensitivity, sodium selectivity, specificity for FMRFamide and Phe-Leu-Arg-Phe-amide (FLRFamide), and no dependency on G-protein coupling. The sodium current also exhibited rapid desensitization in response to repeated FMRFamide applications. Lowering of the pH of the bathing solution reduced the amplitude of the FMRFamide-gated inward current, while also activating an additional sustained weak inward current that was apparently not mediated by the FaNaC. Acidification also prevented the desensitization of the FMRFamide-induced inward current. The acid sensitivity of LsFaNaC is consistent with the hypothesis that FaNaCs share a common ancestry with the ASICs.

Effect of anti-transforming growth factor-beta antibodies in cyclosporine-induced renal dysfunction.

BACKGROUND: Several experimental and clinical studies have implicated a role for transforming growth factor-beta (TGF-beta) in mediating the nephrotoxic effects of cyclosporine (CsA). To test this hypothesis, we administered neutralizing anti-TGF-beta antibodies (alpha-TGF-beta) in a well-described rat model of chronic CsA nephrotoxicity. METHODS: We studied three groups (N = 9 per group) of adult, male Sprague-Dawley rats that received a low-salt diet (0.05% sodium). Normal controls were given vehicle subcutaneously and an alternate-day intraperitoneal injection of 3 mg of nonspecific mouse IgG (MIgG) for 28 days. The CsA group received 15 mg/kg/day of CsA subcutaneously and 3 mg of MIgG intraperitoneally on alternate days for 28 days. The CsA/alpha-TGF-beta group received CsA and alternate-day alpha-TGF-beta (3 mg) for 28 days. At the end of 28 days, creatinine clearance was measured by 24-hour urine collection. Histologic assessment was performed for tubulointerstitial damage and arteriolar hyalinosis. Northern analysis was performed for alpha 1(I) collagen and TGF-beta 1 gene expression, and quantitative reverse transcription-polymerase chain reaction was performed to measure levels of tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-2 (MMP-2), and MMP-9. RESULTS: CsA-treated rats had significantly lower creatinine clearance as compared with normal controls (0.43 +/- 0.07 vs. 0.67 +/- 0.14 mL/min, P = 0.0002), increased interstitial damage and afferent arteriolar hyalinosis (P = 0.0001), and increased alpha1(I) collagen (4-fold) and TGF-beta 1 (2.5-fold) mRNA expression. CsA-treated rats also had significantly increased TIMP-1 (7.4-fold, P < 0.001), MMP-2, and PAI-1 (all approximately 2-fold, P < 0.02) and decreased MMP-9 (85% reduction, P < 0.001) as compared with controls. Treatment with alpha-TGF-beta in CsA-treated rats significantly prevented the reduction in creatinine clearance (0.58 +/- 0.03 mL/min, P = 0.009 vs. CsA alone), the increase in afferent arteriolar hyalinosis (P < 0.05 vs. CsA alone), normalized alpha 1(I) collagen mRNA levels, and attenuated CsA effects on TGF-beta1, TIMP-1, and MMP-9. CONCLUSIONS: In this rat model of CsA-induced nephrotoxicity, renal insufficiency and characteristic histologic changes are associated with altered expression of matrix and matrix-regulating molecules. Based on our results with alpha-TGF-beta antibodies, many but not all of these nephrotoxic effects of CsA are mediated by TGF-beta.

Multimeric CREB-binding sites in the promoter regions of a family of G-protein-coupled receptors related to the vertebrate galanin and nociceptin/orphanin-FQ receptor families.

Four related genes encoding a family of G-protein-coupled receptors (GPCRs) have been isolated from the mollusc Lymnaea stagnalis. The coding regions of this family of receptors share 97-99% sequence similarity at both the protein and nucleotide level, and they also share high sequence identity with vertebrate galanin and orphanin-FQ/nociceptin GPCR families. Analysis of the promoter regions reveals shared domains, some of which encode highly conserved repeating units. One 27-bp repeating unit, which encodes a c-AMP response element (CRE) and binds CREB protein, is repeated 14 times in one promoter. In situ hybridization showed expression of these receptors in identified neurons of several behaviourly important networks including those involved in feeding and ion and water regulation. These Lymnaea receptors are likely to represent members of a novel family of invertebrate neuropeptide receptors extensively regulated in response to intracellular signalling cascades.

Reflections on tissue engineering and trauma therapy.

Although all of Trauma Care has remarkably improved during the latter half of the last century the treatment of burn injury has substantially out paced other areas and serves as an excellent example illustrating the improvements taking place in the treatment of injured patients. Although Trauma treatment lagged behind the rest of medicine in mid-century today it rests solidly on the cutting edge of advancing therapeutic knowledge and practice in areas of metabolism, immunology, infection control, critical care, tissue engineering and the delivery of clinical care. It is important to understand what has happened to allow the treatment of injury to be so effective. The bench mark therapeutic moves that have had major effects allowing the present highly effective treatment are: immediate tailored fluid resuscitation, preventive (prophylactic) and topical antibiotics, metabolically designed nutritional therapy and most important early definitive repair of the injury. All must be delivered early after trauma if they are to be optimally effective in preventing the complications of injury that are devastating if encountered. Unfortunately, all problems are not solved by todays treatment of injury, improved as it is. These problems are largely related to an inexact understanding: of the physiologic changes of aging, of the exact pathophysiologic events in inhalation injury and multisystem organ failure and of the technology required to replace those body parts damaged by the injury itself that lead to death or to healing with loss of function. There will be major improvements in the understanding and ability to effectively deal with the problems of aging and inhalation injury through basic and clinical research but perhaps the major improvement in injury treatment will come through the ability to replace worn out, defective or damaged body parts through technologies that resemble regeneration. Here the concepts of Tissue Engineering have much to contribute and it is worth exploring the donation of Tissue Engineering to dermal replacement following burn injury to serve as an example of what types of additions to treatment Tissue Engineering can make.

Artificial skin.

The skin is a complex organ that is difficult to replace when it is irreversibly damaged by burns, trauma, or disease. Although autologous skin transplantation remains the most common form of treatment in patients with significant skin loss, there are now a number of commercially available products that can be used to replace the skin temporarily or permanently. Here we describe several such products under the rubric "artificial skin," focusing on two types of technology that have been applied to the problem of permanent skin replacement.

Werner's syndrome T lymphocytes display a normal in vitro life-span.

Werner's syndrome (WS) is an autosomal recessive disorder displaying many features consistent with accelerated ageing. Fibroblasts from WS patients show a distinct mutator phenotype (characterised by the production of large chromosomal deletions) and a profound reduction in proliferative capacity. The disorder results from a mutation in a novel ReqQ helicase. Recently, we demonstrated that the proliferative defect was corrected by the ectopic expression of telomerase. From these data, we propose that mutations in the wrn gene lead to deletions at or near the telomere which reduce the cells replicative life-span. This hypothesis predicts that cell types which retain the ability to upregulate telomerase as part of their response to a proliferative stimulus would fail to show any significant effect of wrn gene mutations upon life-span. Human T lymphocytes represent a well-characterised example of such a cell type. To test the hypothesis, WS T lymphocytes were cultured until they reached replicative senescence. These cultures displayed life-spans which did not differ significantly from those of normal controls. These findings are consistent with the hypothesis that the effects of wrn mutations on replicative life-span are telomere-mediated.

Association of IgA nephropathy with Clostridium difficile colitis.

Immunoglobulin A (IgA) nephropathy, the most common cause of glomerulonephritis worldwide, is usually idiopathic in origin and renal limited. Secondary IgA nephropathy has been associated with systemic disease, including such gastrointestinal tract disturbances as celiac sprue and inflammatory bowel disease. We describe gross hematuria and reversible acute renal failure from IgA nephropathy in a patient with cephalosporin-induced Clostridium difficile colitis. In addition to mesangial IgA and C3 deposition, renal histological examination showed glomerular bleeding, intratubular red blood cell casts, and acute tubular necrosis. To the best of our knowledge, this is the first report of an association between IgA nephropathy and C difficile colitis.

A novel human astrocyte cell line (A735) with astrocyte-specific neurotransmitter function.

Studies of brain cell function and physiology are hampered by the limited availability of immortal human brain-derived cell lines, as a result of the technical difficulties encountered in establishing immortal human cells in culture. In this study, we demonstrate the application of recombinant DNA vectors expressing SV40 T antigen for the development of immortal human cell cultures, with morphological, growth, and functional properties of astrocytes. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. One of these cultures gave rise to an immortal cell line, designated A735. All the human SV40-derived lines retained morphological features and growth properties of type 1 astrocytes. Immunohistochemical studies and Western blot analysis of the intermediate filament proteins and glutamine synthetase demonstrated a differentiated but immature astrocyte phenotype. Transport of gamma-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines' retaining aspects of normal glial function. We conclude that methods based on the use of SV40 T antigen can successfully immortalize human astrocytes, retaining key astrocyte functions, but T antigen-induced proliferation appeared to interfere with expression of glial fibrillary acidic protein. We believe A735 is the first documented nontumor-derived human glial cell line which is immortal.