Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids.

Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family is increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically-engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach strengthens our ability to quantify and characterize the inherent diversity of acyl chains across the lipidome.

Cognitive function, quality of life and mental health in survivors of our-of-hospital cardiac arrest: a review.

There is growing interest in the long-term outcomes of patients surviving out-of-hospital cardiac arrest (OHCA). This paper aims to summarise the available literature on the long-term cognitive, health-related quality of life (QoL) and mental health outcomes of survivors of OHCA. Between 30% and 50% of survivors of OHCA experience cognitive deficits for up to several years post-discharge. Deficits of attention, declarative memory, executive function, visuospatial abilities and verbal fluency are commonly reported. Survivors of OHCA appear to report high rates of mental illness, with up to 61% experiencing anxiety, 45% experiencing depression and 27% experiencing post-traumatic stress. Fatigue appears to be a commonly reported long-term outcome for survivors of OHCA. Investigations of long-term QoL for these patients have produced mixed findings. Carers of survivors of OHCA report high rates of depression, anxiety and post-traumatic stress, with insufficient social and financial support. The heterogeneous range of instruments used to assess cognitive function and QoL prevent any clear conclusions being drawn from the available literature. The potential biases inherent in this patient population and the interaction between QoL, cognitive performance and mental health warrant further investigation, as does the role of post-discharge support services in improving long-term patient outcomes.

Battle of the hemichannels--Connexins and Pannexins in ischemic brain injury.

Perinatal ischemic brain injury can occur as a result of a global ischemic insult or focal ischemic stroke in the preterm or full-term neonate. One of the most striking features of HI injury is that, after initial recovery of cellular oxidative metabolism, there is a delayed, 'secondary' mitochondrial failure that spreads over time from the most severely damaged areas outwards, into previously undamaged regions. This secondary failure is accompanied by transient seizure activity and cytotoxic edema. The specific mechanisms of this spread are poorly understood, but it is at least partly associated with spreading waves of depression that can trigger cell death in neighboring uninjured tissues. Both Connexin and Pannexin hemichannels may mediate release of paracrine molecules that in turn propagate cell death messages by releasing intracellular mediators, such as ATP, NAD(+), or glutamate or by abnormally prolonged opening to allow cell edema. This review will discuss the controversy around the relative contribution of both Connexin and Pannexin hemichannels and mechanisms by which they may contribute to the spread of ischemic brain injury.

Connexin hemichannel blockade is neuroprotective after, but not during, global cerebral ischemia in near-term fetal sheep.

There is increasing evidence that connexin hemichannels, the half gap junctions that sit unopposed in the cell membrane, can open during ischemia and that blockade of connexin43 hemichannels after cerebral ischemia can improve neural outcomes. However, it is unclear whether connexin blockade during ischemia is protective. In the present study global cerebral ischemia was induced by 30 min of bilateral carotid artery occlusion in near-term (128 ± 1 day gestation age) fetal sheep. A specific mimetic peptide that blocks connexin43 hemichannels was infused into the lateral ventricle for either 1h before and during ischemia (intra-ischemia group, n=6) or for 25 h starting 90 min after the end of ischemia (post-ischemia group, n=7). The vehicle was infused in the ischemia-vehicle group (n=6) and sham-controls received sham occlusion plus vehicle (n=10). The post-ischemia group showed enhanced recovery of EEG power from day five until the end of the experiment (-5 ± 1.6 dB) compared to ischemia-vehicle (-13 ± 1.9 dB, p<0.05) and intra-ischemia infusion (-14.4 ± 3.6 dB, p<0.05). Post-ischemic infusion was associated with higher neuronal counts compared to ischemia-vehicle and intra-ischemia in the cortex (p<0.05) but not the CA1 and CA3 regions of the hippocampus. Oligodendrocyte cell counts in the intragyral and periventricular white matter were significantly higher in the post-ischemia group compared to ischemia-vehicle and intra-ischemia infusion (p<0.05). These large animal data support the hypothesis that connexin hemichannel opening after, but not during, ischemia contributes to the spread of white and gray matter injury of the developing brain.

In-vitro and in-vivo evaluation of carrageenan/methylcellulose polymeric systems for transscleral delivery of macromolecules.

In this study, polymeric dispersions composed of methylcellulose (MC) and either kappa carrageenan (KC) or iota carrageenan (IC) were proposed as a platform for transscleral delivery of macromolecules. The additive effects of the two polymers were investigated using oscillatory rheometer and FT-IR spectroscopy. Mechanical spectra demonstrated a conformation dependent association of the two polymers at 37 °C in the presence of selected counter ions. The polymer association was also confirmed by the shifts in MC peaks at 1049.5, 1114 and 1132.9 cm(-1) in the presence of carrageenans, which corresponds to the stretching vibrations of C-O-C bonds of the polysaccharides. The MC-IC polymeric system displayed the highest bio-adhesion, owing to the relatively high negative charge. However, the MC-IC system did not affect the in-vitro scleral permeability of sodium fluorescein and 10 kDa FITC-dextran. Nonetheless, the formulation properties had a substantial impact on the results of the in-vivo studies. The efficacy of transscleral drug delivery was determined using rats with altered connexin 43 (Cx43) levels, a gap junction protein, in the choroid. Periocular injection of Cx43 antisense oligonucleotides (AsODN) incorporated in the MC-IC system lead to a significant reduction in the Cx43 levels in the choroid of rats at 24 h of treatment. AsODN incorporated in phosphate buffered saline (PBS) also demonstrated a trend towards reduced Cx43 levels; however this was not statistically significant owing to great variability between treated animals. Consequently the in-vivo data suggests the transscleral route to be of value in delivering therapeutics to the choroid. Moreover this study identified a new polymeric system based on MC and IC which provides aqueous loading of therapeutics and prolonged retention at the site of administration.

Antisense down regulation of connexin31.1 reduces apoptosis and increases thickness of human and animal corneal epithelia.

The roles of the gap junction protein connexin31.1 (Cx31.1) are poorly understood, especially as the protein appears to form non-functional channels. Cx31.1 specific antisense oligodeoxynucleotides (ODNs) were designed to evaluate its roles in a corneal epithelium model. Expression of Cx31.1 in corneal epithelium extends from the suprabasal layers of polyhedral wing cells through to the flat squamous cells of superficial layers which are shed into the tear film. Deoxyribozymes (Dzs) were tested for cleavage efficacy using in vitro transcribed Cx31.1 mRNA. Cleavage results showed a putative tertiary structure for Cx31.1 mRNA with one region appearing to have a higher potential for antisense targeting. Application of antisense ODNs designed to this region caused Cx31.1 knockdown in rat and human corneal organotypic culture models, leading to a reduction in apoptosis and a thickening of the corneal epithelium (p=0.0045). Cx31.1 appears to play a role in triggering cell death; knocking it down may provide a novel approach for tissue repair and engineering.

Oculomotor control in children with fetal alcohol spectrum disorders assessed using a mobile eye-tracking laboratory.

Prenatal exposure to alcohol can result in a spectrum of adverse developmental outcomes, collectively termed fetal alcohol spectrum disorders (FASDs). This study evaluated deficits in sensory, motor and cognitive processing in children with FASD that can be identified using eye movement testing. Our study group was composed of 89 children aged 8-15 years with a diagnosis within the FASD spectrum [i.e. fetal alcohol syndrome (FAS), partial fetal alcohol syndrome (pFAS), and alcohol-related neurodevelopmental disorder (ARND)], and 92 controls. Subjects looked either towards (prosaccade) or away from (antisaccade) a peripheral target that appeared on a computer monitor, and eye movements were recorded with a mobile, video-based eye tracker. We hypothesized that: (i) differences in the magnitude of deficits in eye movement control exist across the three diagnostic subgroups; and (ii) children with FASD display a developmental delay in oculomotor control. Children with FASD had increased saccadic reaction times (SRTs), increased intra-subject variability in SRTs, and increased direction errors in both the prosaccade and antisaccade tasks. Although development was associated with improvements across tasks, children with FASD failed to achieve age-matched control levels of performance at any of the ages tested. Moreover, children with ARND had faster SRTs and made fewer direction errors in the antisaccade task than children with pFAS or FAS, although all subgroups were different from controls. Our results demonstrate that eye tracking can be used as an objective measure of brain injury in FASD, revealing behavioral deficits in all three diagnostic subgroups independent of facial dysmorphology.

Executive function deficits in children with fetal alcohol spectrum disorders (FASD) measured using the Cambridge Neuropsychological Tests Automated Battery (CANTAB).

BACKGROUND: Chronic prenatal alcohol exposure causes a spectrum of deleterious effects in offspring, collectively termed fetal alcohol spectrum disorders (FASD), and deficits in executive function are prevalent in FASD. The goal of this research was to test the hypothesis that children with FASD exhibit performance deficits in tasks that assess attention, planning and spatial working memory. METHODS: Subjects (8-15 years male and female children) with a diagnosis of fetal alcohol syndrome (FAS), partial FAS (pFAS), or alcohol-related neurodevelopmental disorder (ARND), and age- and sex-matched controls, completed four tasks selected from the Cambridge Neuropsychological Tests Automated Battery (CANTAB). RESULTS: Compared with age-matched control children (n = 92), subjects with FASD (n = 89) exhibited longer reaction and decision times (effect size range; Cohen's d = .51 to .73), suggesting deficits in attention. Children with FASD demonstrated deficits in planning and spatial working memory that became more pronounced when task difficulty increased. The largest effect size in this study population (Cohen's d = 1.1) occurred in the spatial working memory task. Only one outcome measure revealed differences across the diagnostic subgroups, although all groups were different from control. CONCLUSION: This study demonstrates that deficits in multiple executive function domains, including set shifting, planning and strategy use, attention and spatial working memory, can be assessed in children with FASD using an easy to administer, brief battery of computer-based neuropsychological tasks. The tasks appear to be equally sensitive for brain injury resulting from prenatal exposure to alcohol, regardless of the presence of facial dysmorphology.

Effects of chronic prenatal ethanol exposure on mitochondrial glutathione and 8-iso-prostaglandin F2alpha concentrations in the hippocampus of the perinatal guinea pig.

It is hypothesised that oxidative stress is a key mechanism of ethanol neurobehavioural teratogenicity, resulting in altered endogenous antioxidant status and increased membrane lipid peroxidation in the hippocampus of chronic prenatal ethanol exposure (CPEE) offspring. To test this hypothesis, timed pregnant guinea-pigs (term, approximately gestational day (GD) 68) received chronic daily oral administration of (i) 4 g ethanol kg(-1) maternal bodyweight, (ii) isocaloric sucrose with pair feeding, or (iii) water. At GD 65 (term fetus) and postnatal day (PD) 0 (neonate), individual offspring were killed, the brain was excised and the hippocampi were dissected. Glutathione (GSH) concentration was measured in the cytosolic and mitochondrial fractions of hippocampal homogenate. The occurrence of lipid peroxidation was determined by measuring the concentration of 8-iso-prostaglandin F2+/- (8-iso-PGF2+/-). There was CPEE-induced decreased brain weight and hippocampal weight at GD 65 and PD 0, decreased mitochondrial GSH concentration in the hippocampus at PD 0, with no change in mitochondrial GSH concentration at GD 65 or cytosolic GSH concentration at GD 65 or PD 0, and no change in mitochondrial or whole-homogenate 8-iso-PGF2+/- concentration in the hippocampus at GD 65 or PD 0. The data demonstrate that CPEE produces selective mitochondrial dysfunction in the hippocampus of the neonatal guinea-pig, involving GSH depletion.

Chronic prenatal ethanol exposure increases apoptosis in the hippocampus of the term fetal guinea pig.

It is hypothesized that chronic prenatal ethanol exposure (CPEE), via maternal ethanol administration, increases mitochondrial-directed apoptosis in the hippocampus of the term fetus that precedes loss of hippocampal CA1 pyramidal cells. To test this hypothesis, timed pregnant guinea pigs received chronic oral administration of: 4 g ethanol/kg maternal body weight/day, isocaloric-sucrose/pair-feeding or water throughout gestation. At gestational day 65 (term fetus) and postnatal day 0 (neonate), individual offspring were euthanized, and the brain was excised and dissected. CPEE, compared with the isocaloric-sucrose/pair-fed and water control groups, decreased the brain weight of the term fetus and neonate. CPEE did not alter the density of CA1 pyramidal cells in the hippocampus of the term fetus and neonate. In the term fetus, CPEE increased cytochrome c content in the cytosolic fraction of the hippocampus, altered the mitochondrial localization of cytochrome c in cells of the dorsal hippocampus, and increased the percentage of cells in the dorsal hippocampus containing activated caspase-3 and cleaved poly(ADP-ribose) polymerase. The data indicate that CPEE increases neuroapoptosis in the hippocampus of term fetus, which appears to occur via an intrinsic, mitochondrial-directed mechanism initiated by leakage of pro-apoptotic cytochrome c from mitochondria into the cytoplasm.

Limiting burn extension by transient inhibition of Connexin43 expression at the site of injury.

Extension of a burn wound over the first 24h following injury is recognised clinically, and leads to diagnostic and therapeutic dilemmas. In the central nervous system, a similar spread of damage, beyond the initial injury, can occur via the spread of death signals from injured cells to their healthy neighbours via Connexin43 (Cx43) gap junction channels. In the skin, Cx43 is expressed in the basal epidermis and in fibroblasts and dermal appendages. We have used Cx43 specific antisense oligodeoxynucleotide approach to transiently down-regulate Cx43 protein in the early stages of partial thickness cutaneous burn wound healing. Antisense ODNs reduce the spread of tissue damage and neutrophil infiltration around the wound following injury. Epithelial cell proliferation is increased and the rate of wound closure is accelerated, compared to controls. Resultant scarring is smaller with less granulation tissue and more dermal appendages than controls. These findings suggest that Cx43 antisense treatment speeds partial thickness burn wound healing and reduces scarring. We suggest that this approach may provide an effective adjunct to managing partial thickness burn wounds.

Connexin43 gap junction protein plays an essential role in morphogenesis of the embryonic chick face.

Normal outgrowth and fusion of facial primordia during vertebrate development require interaction of diverse tissues and co-ordination of many different signalling pathways. Gap junction channels, made up of subunits consisting of connexin proteins, facilitate communication between cells and are implicated in embryonic development. Here we describe the distribution of connexin43 and connexin32 gap junction proteins in the developing chick face. To test the function of connexin43 protein, we applied antisense oligodeoxynucleotides that specifically reduced levels of connexin43 protein in cells of early chick facial primordia. This resulted in stunting of primordia outgrowth and led to facial defects. Furthermore, cell proliferation in regions of facial primordia that normally express high levels of connexin43 protein was reduced and this was associated with lower levels of Msx-1 expression. Facial defects arise when retinoic acid is applied to the face of chick embryos at later stages. This treatment also resulted in significant reduction in connexin43 protein, while connexin32 protein expression was unaffected. Taken together, these results indicate that connexin43 plays an essential role during early morphogenesis and subsequent outgrowth of the developing chick face.

Detection of submicroscopic magnetite particles using reflectance mode confocal laser scanning microscopy.

Light microscopy and transmission electron microscopy work at such different scales that some components of cells may be too small to detect using light microscopy but too dispersed among cells within tissues to be discovered using electron microscopy. We have used reflectance mode confocal laser scanning microscopy to detect single-domain magnetite crystals in both live and resin-embedded preparations of magnetotactic bacteria. We show that reflections from bacterial cells are uniquely associated with the magnetite, which underpins the magnetotactic response of the bacteria. En bloc viewing shows that relatively large volumes of material can be searched with sufficient resolution to enable detection of submicroscopic particles. The techniques reported here may be of interest to others wishing to detect submicroscopic objects dispersed in large volumes of tissue.

Resolution of refractory symptoms of secondary erythermalgia with intermittent epidural bupivacaine.

BACKGROUND AND OBJECTIVES: Erythermalgia is a rare disorder characterized by erythema, edema, elevated skin temperature, and burning pain of the hands and/or feet. The etiology of primary erythermalgia is idiopathic while secondary erythermalgia is associated with autoimmune and rheumatologic factors. Symptoms are typically refractory to medical management. We report the use of epidural bupivacaine as a treatment option for secondary erythermalgia. CASE REPORT: A 28-year-old woman presented with secondary erythromelalgia that was characterized by severe pain, swelling, and erythema. Medical management failed and a lumbar epidural dosed with bupivacaine was used. This resulted in complete resolution of the patient's symptoms. CONCLUSION: Intermittent epidural bupivacaine may provide treatment of the refractory symptoms of secondary erythermalgia.

Analysis of the physician variable in pain management.

BACKGROUND: The role of physician variability in pain management is unknown. OBJECTIVE: To assess the role of physician variability in the management of pain and provide quantitative data regarding the status of pain management in Michigan. DESIGN: A multi-item mail survey was used to determine the physician's perceived knowledge of pain management modalities, goals, satisfaction, and confidence with pain treatment. Participants. The focus of this report was a group of 368 licensed Michigan physicians who provide clinical care. RESULTS: Overall, 30% of the study group reported no formal education in pain management, although younger physicians reported more education (correlation coefficient = -0.252, P <.001). The physicians reported greater confidence in their knowledge of meperidine than other Schedule II opioids (P <.001 ). In regards to the opinion that prescribing strong opioids would attract a medical review, the physician responses ranged from 1 (strongly disagree) to 5 (strongly agree). The median score for this scale was 4, accounting for 46% of the responses. The study group expressed less satisfaction with their treatment of chronic pain as well as lower goals for relief (mean: 3.8; 95% confidence interval: 3.7-3.9). CONCLUSIONS: Lower expectations for relief and less satisfaction in its management may contribute to the undertreatment of chronic pain. Perceptions of regulatory scrutiny may contribute to suboptimal pain management. These preliminary data highlight physician variability in pain decision making while providing insights into educational needs.

The role of childhood and adulthood abuse among women presenting for chronic pain management.

OBJECTIVE: This study investigated the association between repeated childhood and adulthood abuse and somatic symptom reporting, mental health care use, and substance use among women with chronic pain. DESIGN: A survey of a consecutive sample. PATIENTS: Ninety consecutive women patients presenting for chronic pain management at a multidisciplinary pain management center. OUTCOME MEASURES: The authors assessed the presence or absence of physical or sexual abuse (using the Drossman Physical-Sexual Abuse Survey), period of abuse, demographics, mental health care use, drug or alcohol use and substance abuse, and the presence or absence of physical, pain, and anxiety (somatic) symptoms. RESULTS: The response rate among patients surveyed was 64%. Of the 43 respondents (48%) who reported abuse, 17 (40%) cited childhood abuse, 12 (28%) cited adulthood abuse, and 14 (33%) cited repeated abuse. Women describing long-term abuse reported a significantly greater number of physical, pain, and anxiety symptoms and were more likely to report a history of substance abuse than women reporting abuse during childhood or adulthood alone. CONCLUSIONS: These data indicate a significant association between health status and reported abuse among women presenting to a multidisciplinary pain center for pain management. This finding is consistent with those of previous investigators, and emphasizes the importance of routine evaluation of the presence of long-term abuse as a possible predictor of the onset of chronic pain states.

Use of the McGill Pain Questionnaire to compare women with vulvar pain, pelvic pain and headaches.

OBJECTIVE: To assess differences between women with three distinct types of chronic pain conditions using a modified McGill Pain Questionnaire. STUDY DESIGN: Data by self-administered questionnaire were collected on patients presenting to the University of Michigan Medical Center with chronic vulvar pain (144 patients), pelvic pain (198 patients) or headaches (130 patients). Data for analysis included: patient demographics, duration of pain and modified McGill Pain Questionnaire scores. Univariate and multivariate analyses were performed. RESULTS: Patients with vulvar pain had more formal education (P < .001), were more likely to be married (P < .001) and were less likely to be African American (P = .003) as compared to those with chronic pelvic pain and headaches. Chronic pelvic pain patients were younger than those in the other two groups (P = .002), and headache patients were likely to have had their chronic pain for a shorter duration than those with vulvar or pelvic pain (P < .001). Patients with vulvar pain had lower total scores on the McGill Pain Questionnaire as well as on the four subsets of variables: affective, sensory, cognitive and miscellaneous indexes (P < .001). They also chose fewer words to describe their symptoms from the 20-word lists (P < .001) and had lower average scores in each of the 20 categories as compared to the other two groups (P < .0001). Controlling for age, ethnicity and marital status did not alter this significance. CONCLUSION: Patients with vulvar pain were a unique groups when compared to other chronic pain populations. Evaluation of the demographics and McGill Pain Questionnaire scores confirmed the distinct qualities of women with vulvar pain.

Magnetite defines a vertebrate magnetoreceptor.

The key behavioural, physiological and anatomical components of a magnetite-based magnetic sense have been demonstrated in rainbow trout (Oncorhynchus mykiss). Candidate receptor cells located within a discrete sub-layer of the olfactory lamellae contained iron-rich crystals that were similar in size and shape to magnetite crystals extracted from salmon. Here we show that these crystals, which mapped to individual receptors using confocal and atomic force microscopy, are magnetic, as they are uniquely associated with dipoles detected by magnetic force microscopy. Analysis of their magnetic properties identifies the crystals as single-domain magnetite. In addition, three-dimensional reconstruction of the candidate receptors using confocal and atomic force microscopy imaging confirm that several magnetic crystals are arranged in a chain of about 1 microm within the receptor, and that the receptor is a multi-lobed single cell. These results are consistent with a magnetite-based detection mechanism, as 1-microm chains of single-domain magnetite crystals are highly suitable for the behavioural and physiological responses to magnetic intensity previously reported in the trout.

Calciphylaxis treated with neurolytic lumbar sympathetic block: case report and review of the literature.

BACKGROUND AND OBJECTIVE: Calciphylaxis is a painful complication of end-stage renal disease and secondary hyperparathyroidism. Calcification most commonly affects skin and soft tissue of the lower extremities resulting in excruciatingly painful skin ulcers. Treatment involves correction of hypercalcemia and hyperphosphatemia, parathyroidectomy, and supportive measures. METHODS: The literature and the merits of neurolytic lumbar sympathetic blockade (LSB) for the treatment of pain associated with calciphylaxis are reviewed. CONCLUSIONS: The neurolytic LSB provided pain relief and is a treatment modality to be considered in managing the pain associated with calciphylaxis.