FOXO3a as a sensor of unilateral nerve injury in sensory neurons ipsilateral, contralateral and remote to injury.

Emerging evidence supports that the stress response to peripheral nerve injury extends beyond the injured neuron, with alterations in associated transcription factors detected both locally and remote to the lesion. Stress-induced nuclear translocation of the transcription factor forkhead class box O3a (FOXO3a) was initially linked to activation of apoptotic genes in many neuronal subtypes. However, a more complex role of FOXO3a has been suggested in the injury response of sensory neurons, with the injured neuron expressing less FOXO3a. To elucidate this response and test whether non-injured sensory neurons also alter FOXO3a expression, the temporal impact of chronic unilateral L4-6 spinal nerve transection on FOXO3a expression and nuclear localization in adult rat dorsal root ganglion neurons ipsilateral, contralateral or remote to injury relative to naïve controls was examined. In naïve neurons, high cytoplasmic and nuclear levels of FOXO3a colocalized with calcitonin gene related peptide, a marker of the nociceptive subpopulation. One hour post-injury, an acute increase in nuclear FOXO3a in small size injured neurons occurred followed by a significant decrease after 1, 2 and 4 days, with levels increasing toward pre-injury levels by 1 week post-injury. A more robust biphasic response to the injury was observed in uninjured neurons contralateral to and those remote to injury. Nuclear levels of FOXO3a peaked at 1 day, decreased by 4 days, then increased by 1 week post-injury, a response mirrored in C4 dorsal root ganglion neurons remote to injury. This altered expression contralateral and remote to injury supports that spinal nerve damage has broader systemic impacts, a response we recently reported for another stress transcription factor, Luman/CREB3. The early decreased expression and nuclear localization of FOXO3a in the injured neuron implicate these changes in the cell body response to injury that may be protective. Finally, the broader systemic changes support the existence of stress/injury-induced humeral factor(s) influencing transcriptional and potentially behavioral changes in uninjured dorsal root ganglion neurons. Approval to conduct this study was obtained from the University of Saskatchewan Animal Research Ethics Board (protocol #19920164).

Is there a pulse wave encephalopathy component to multiple sclerosis?

The dominant hypothesis in multiple sclerosis is that it is an autoimmune disease; however, there is considerable evidence that the immune attack on myelin may be secondary to a cytodegenerative event. Furthermore, the immune modulating therapies longest in clinical use, although modulating the frequency and severity of exacerbation, do not affect long-term progression towards disability. Clearly alternative perspectives on the etiology of multiple sclerosis are warranted. In this paper I outline the commonalities between idiopathic normal pressure hydrocephalus and multiple sclerosis. These include decreased intracranial compliance as evidenced by increased cerebrospinal fluid volume and velocity of cerebrospinal fluid flow through the cerebral aqueduct; increased ventricular volume; periventricular demyelination lesions; increase in size of Virchow-Robin spaces; presence of Hakim's triad comprised of locomotory disabilities, cognitive problems and bladder control problems. Furthermore, multiple sclerosis is associated with decreased arterial compliance. These are all suggestive that there is a pulse wave encephalopathy component to multiple sclerosis. There are enough resemblances between normal pressure hydrocephalus and multiple sclerosis to warrant further investigation. Whether decreases in intracranial compliance is a consequence of multiple sclerosis or is a causal factor is unknown. Effective therapies can only be developed when the etiology of the disease is understood.

Hydroxybenzoic acid isomers and the cardiovascular system.

Today we are beginning to understand how phytochemicals can influence metabolism, cellular signaling and gene expression. The hydroxybenzoic acids are related to salicylic acid and salicin, the first compounds isolated that have a pharmacological activity. In this review we examine how a number of hydroxyphenolics have the potential to ameliorate cardiovascular problems related to aging such as hypertension, atherosclerosis and dyslipidemia. The compounds focused upon include 2,3-dihydroxybenzoic acid (Pyrocatechuic acid), 2,5-dihydroxybenzoic acid (Gentisic acid), 3,4-dihydroxybenzoic acid (Protocatechuic acid), 3,5-dihydroxybenzoic acid (α-Resorcylic acid) and 3-monohydroxybenzoic acid. The latter two compounds activate the hydroxycarboxylic acid receptors with a consequence there is a reduction in adipocyte lipolysis with potential improvements of blood lipid profiles. Several of the other compounds can activate the Nrf2 signaling pathway that increases the expression of antioxidant enzymes, thereby decreasing oxidative stress and associated problems such as endothelial dysfunction that leads to hypertension as well as decreasing generalized inflammation that can lead to problems such as atherosclerosis. It has been known for many years that increased consumption of fruits and vegetables promotes health. We are beginning to understand how specific phytochemicals are responsible for such therapeutic effects. Hippocrates' dictum of 'Let food be your medicine and medicine your food' can now be experimentally tested and the results of such experiments will enhance the ability of nutritionists to devise specific health-promoting diets.

The evidence for hypoperfusion as a factor in multiple sclerosis lesion development.

The evidence that hypoxia is a precipitating factor in causing early MS lesions includes increased protein levels of hypoxia-inducible factor-1 α ; presence of the D-110 hypoxia-inducible protein; increased expression of hypoxia-inducible genes in lesions as well as in adjacent normal-appearing white matter (NAWM); loss of myelin-associated glycoprotein in myelin of early MS lesions; a 50% reduction of blood flow through NAWM with areas of lowest blood flow having the greatest probability of lesion development. Why MS-like lesions develop following hypoxemic insults in some individuals but not in others is likely dependent upon the presence of immune predisposing factors that are governed genetically. Hypoperfusion may be due to decreased arterial supply, restricted venous return, or a combination of these. There are clinical trials ongoing or planned to treat chronic cerebrospinal venous insufficiency (CCSVI) through angioplasty. I suggest that it is important that clinical trials addressing vascular issues in MS should examine how the vascular intervention affects white matter perfusion and determine whether the extent of perfusion recovery and maintenance of this recovery is related to functional recovery and maintenance of functional recovery. Consideration should also be given to the possibility of arterial problems playing a role in hypoperfusion in some MS patients.

In vivo visualization of gold-loaded cells in mice using x-ray computed tomography.

The ability to perform cell tracking using x-ray computed tomography combined with gold nanoparticles has been demonstrated recently on ex vivo samples using different malignant and nonmalignant cell lines. Here we proved the concept of the method for in vivo assessment in a small-animal model of malignant brain tumors. The limitations of the method due to radiation dose constraints were investigated using Monte Carlo simulations. Taking into consideration different x-ray entrance doses and the spatial resolution, the visibility of the cell clusters was evaluated. The results of the experiments conducted on mice implanted with F98 tumor cells confirmed the prediction of the Monte Carlo calculations. Small clusters of cells exogenously loaded with gold nanoparticles could be visualized using our in vivo method. FROM THE CLINICAL EDITOR: This article discusses the use of CT-based detection of gold nanoparticle loaded cells of interest in small-animal models of malignant brain tumors, where small clusters of cells loaded with gold nanoparticles could be visualized.

Dietary Nrf2 activators inhibit atherogenic processes.

Dietary Nrf2 activators increase expression of phase 2 protein genes in cells undergoing oxidative stress resulting in a lowering of oxidative stress. Oxidative stress promotes atherogenic processes through oxidizing low density lipoproteins and promotion of inflammation through activation of nuclear factor kappa B and activation of mitogen-activated protein kinases. Nrf2 activators by decreasing oxidative stress decrease the probability of developing atherosclerotic lesions.

How the dissection laboratory facilitates integration of learning: presence of abdominal aortic aneurysm with a large intracardiac thrombus: a rare cadaver finding.

There is a debate among medical education experts on the application of dissection or prosection for learning anatomy. However, the literature reveals that the majority of published articles are in favor of dissection. In this article, we present a case of an abdominal aortic aneurysm (AAA) with intracardiac thrombus in a cadaver on routine dissection. We will discuss possible explanations for such finding and provide some insight into how this finding can support the significance of the cadaver-based teaching of anatomy of the medical students. Initially, the abdomen was dissected and exposed to study the abdominal structures in an anatomy class and later the thoracic region was dissected and all the clinical abnormalities were examined and documented. Autopsy of the clot was obtained for histopathology analysis. The intracardiac thrombus was present in the right atrium characterized by its projection into the superior vena cava, inferior vena cava, and the right ventricle. The AAA was extensive and inferior to the renal arteries constricting the entire inferior vena cava. Moreover, associated findings included presence of numerous collaterals in the thoracic region near the superior vena cava; histological examination of the clot showed extensive population of leukocytes. There were enlarged mediastinal lymph nodes. Our cadaver showed an excellent model for integrating between clinical anatomy and pathology by triggering medical students to think of normal and abnormal structures: often called "thinking outside the box." Such an effort might help them in developing their thought processes and future medical careers.

The dietary phase 2 protein inducer sulforaphane can normalize the kidney epigenome and improve blood pressure in hypertensive rats.

BACKGROUND: Our previous studies have shown that broccoli sprouts high in the glucosinolate glucoraphanin decreases renal and vascular oxidative stress and inflammation as well as blood pressure in spontaneously hypertensive stroke-prone (SHRSP) rats. The objective of this study was to determine whether the metabolite of glucoraphanin, sulforaphane, was responsible for this improved blood pressure and whether this is associated with normalization of renal methylated DNA. METHODS: Sulforaphane was given by gavage to SHRSP and Sprague Dawley (SD) rats over 4 months and blood pressure measured under anesthesia just before euthanasia. Renovascular morphology was determined by histology and methylated deoxycytosine levels analyzed using high-performance liquid chromatography. RESULTS: Mean arterial pressure was 20% higher in vehicle-treated SHRSP when compared to SD. Sulforaphane administration to SHRSP improved blood pressure and lowered this difference to 11%. Vehicle-treated SHRSP had significantly increased wall:lumen ratios in renal arteries, increased numbers of vascular smooth muscle cells (VSMCs), increased renal protein nitration, and decreased (11%) renal DNA methylation compared to SD. Sulforaphane administration to SHRSP significantly lowered arterial wall:lumen ratio by 35%, reduced the number of VSMCs, reduced the level of protein nitration, and increased methylated deoxycytosine levels by 14%. CONCLUSIONS: Sulforaphane administration rectified pathological abnormalities in SHRSP kidneys and significantly improved blood pressure. This was associated with normalization of global kidney DNA methylation suggesting that DNA methylation could be associated with hypertension.

The effect of spinal manipulation on imbalances in leg strength.

We hypothesized that spinal manipulation (SM) would reduce strength imbalances between legs. Using an un-blinded randomized design, 28 males and 21 females (54 ± 19y) with at least a 15% difference in isometric strength between legs for hip flexion, extension, abduction, or knee flexion were randomized to treatment or placebo (mock spinal manipulation). Strength of the stronger and weaker legs for hip flexion, extension, abduction, and/or knee flexion was assessed before and after the intervention. SM reduced the relative strength difference between legs for knee flexion (mean ± SD 57 ± 53 to 5 ± 14%) and hip flexion (24 ± 12 to 11 ± 15%) compared to placebo (34 ± 29 to 24 ± 36%, and 20 ± 18 to 22 ± 26%, respectively) (p = 0.05). SM also improved strength in the weak leg for hip abduction (104 ± 43 to 116 ± 43 Nm) compared to placebo (84 ± 24 to 85 ± 31 Nm) (p = 0.03). This study suggests that spinal manipulation may reduce imbalances in strength between legs for knee and hip flexion.

Gold nanoparticle labeling of cells is a sensitive method to investigate cell distribution and migration in animal models of human disease.

The ability to track cells in small-animal models of human disease is important because it gives the potential to improve our understanding of the processes of disease progression as well as our understanding of the therapeutic effects of interventions. In this study gold nanoparticles have been used as a permanent marker of implanted normal and malignant cell grafts in combination with a suitable x-ray apparatus. Using x-ray computed tomography the micrometric three-dimensional distribution of these marked cells could be displayed with penetration depth, high cell sensitivity and high spatial resolution in rodent models of human diseases. In principle the method allows quantification of cell numbers at any anatomical location over time in small animals.

Health effects with consumption of the flax lignan secoisolariciresinol diglucoside.

Flaxseed is the richest source of the lignan secoisolariciresinol diglucoside (SDG). After ingestion, SDG is converted to secoisolariciresinol, which is further metabolised to the mammalian lignans enterodiol and enterolactone. A growing body of evidence suggests that SDG metabolites may provide health benefits due to their weak oestrogenic or anti-oestrogenic effects, antioxidant activity, ability to induce phase 2 proteins and/or inhibit the activity of certain enzymes, or by mechanisms yet unidentified. Human and animal studies identify the benefits of SDG consumption. SDG metabolites may protect against CVD and the metabolic syndrome by reducing lipid and glucose concentrations, lowering blood pressure, and decreasing oxidative stress and inflammation. Flax lignans may also reduce cancer risk by preventing pre-cancerous cellular changes and by reducing angiogenesis and metastasis. Thus, dietary SDG has the potential to decrease the incidence of several chronic diseases that result in significant morbidity and mortality in industrialised countries. The available literature, though, makes it difficult to clearly identify SDG health effects because of the wide variability in study methods. However, the current evidence suggests that a dose of at least 500 mg SDG/d for approximately 8 weeks is needed to observe positive effects on cardiovascular risk factors in human patients. Flaxseed and its lignan extracts appear to be safe for most adult populations, though animal studies suggest that pregnant women should limit their exposure. The present review discusses the potential health benefits of SDG in humans, with supporting evidence from animal studies, and offers suggestions for future research.

Troglitazone reverses the multiple drug resistance phenotype in cancer cells.

A major problem in treating cancer is the development of drug resistance. We previously demonstrated doxorubicin (DOX) resistance in K562 human leukemia cells that was associated with upregulation of glyoxalase 1 (GLO-1) and histone H3 expression. The thiazolidinedione troglitazone (TRG) downregulated GLO-1 expression and further upregulated histone H3 expression and post-translational modifications in these cells, leading to a regained sensitivity to DOX. Given the pleiotropic effects of epigenetic changes in cancer development, we hypothesized that TRG may downregulate the multiple drug resistance (MDR) phenotype in a variety of cancer cells. To test this, MCF7 human breast cancer cells and K562 cells were cultured in the presence of low-dose DOX to establish DOX-resistant cell lines (K562/DOX and MCF7/DOX). The MDR phenotype was confirmed by Western blot analysis of the 170 kDa P-glycoprotein (Pgp) drug efflux pump multiple drug resistance protein 1 (MDR-1), and the breast cancer resistance protein (BCRP). TRG markedly decreased expression of both MDR-1 and BCRP in these cells, resulting in sensitivity to DOX. Silencing of MDR-1 expression also sensitized MCF7/DOX cells to DOX. Use of the specific and irreversible peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor GW9662 in the nanomolar range not only demonstrated that the action of TRG on MCF/DOX was PPARgamma-independent, but indicated that PPARgamma may play a role in the MDR phenotype, which is antagonized by TRG. We conclude that TRG is potentially a useful adjunct therapy in chemoresistant cancers.

Phase 2 protein inducers in the diet promote healthier aging.

Oxidative stress drives many aging-associated problems. Because oxidative stress can be decreased by induction of phase 2 proteins, we hypothesized that incorporating the phase 2 protein inducer 2(3)-tert-butyl-4-hydroxyanisole (tBHA) into the diet would result in healthier aging. C57BL/6 mice were placed either on control mouse chow diet or on chow containing tBHA and were examined at 6, 12, and 18 months. Dietary tBHA resulted in the antioxidant response activation, decreased both oxidative stress and pro-inflammatory gene expression in tissues examined, counteracted the decrease in the transcription factors peroxisome proliferator-activated receptor-gamma and increase in CCAAT/enhancer binding protein-alpha levels seen in liver with aging, and was associated with mice having less weight gain, despite having no differences in food consumption, and better locomotor function. We conclude that simple changes in the diet such as incorporation of phase 2 protein inducers can have a profound influence on health and, thereby, the aging process.

Memory and survival after microbeam radiation therapy.

BACKGROUND: Disturbances of memory function are frequently observed in patients with malignant brain tumours and as adverse effects after radiotherapy to the brain. Experiments in small animal models of malignant brain tumour using synchrotron-based microbeam radiation therapy (MRT) have shown a promising prolongation of survival times. MATERIALS AND METHODS: Two animal models of malignant brain tumour were used to study survival and memory development after MRT. Thirteen days after implantation of tumour cells, animals were submitted to MRT either with or without adjuvant therapy (buthionine-SR-sulfoximine=BSO or glutamine). We used two orthogonal 1-cm wide arrays of 50 microplanar quasiparallel microbeams of 25 microm width and a center-to-center distance of about 200 microm, created by a multislit collimator, with a skin entrance dose of 350 Gy for each direction. Object recognition tests were performed at day 13 after tumour cell implantation and in monthly intervals up to 1 year after tumour cell implantation. RESULTS: In both animal models, MRT with and without adjuvant therapy significantly increased survival times. BSO had detrimental effects on memory function early after therapy, while administration of glutamine resulted in improved memory.

Diffraction-enhanced imaging of a porcine eye.

BACKGROUND: Diffraction-enhanced imaging (DEI) is a synchrotron-based x-ray imaging technique that has dramatically improved contrast over standard x-ray imaging techniques. It is possible to acquire images that analyze the x-ray refraction and the apparent absorption (elimination of small-angle scattering) of the object. METHODS: Three formalin-fixed porcine eyes were studied at the National Synchrotron Light Source using DEI. Conventional absorption-type radiography was conducted for comparison. RESULTS: Conventional absorption radiography did not yield significant detail of the eye anatomy. DEI showed excellent characterization of many ocular structures. The cornea, iris, lens, retina, optic nerve, as well as choroidal vasculature and the ampullae of the vortex veins could all be visualized. INTERPRETATION: DEI represents a novel, high-resolution imaging technique that has excellent characterization of ocular anatomy. Further application of this imaging modality will be undertaken to study cataract and choroidal tumors and to examine ocular surface structures, such as the extraocular muscle insertions, more closely.

Synchrotron-based intravenous cerebral angiography in a small animal model.

K-edge digital subtraction angiography (KEDSA), a recently developed synchrotron-based technique, utilizes monochromatic radiation and allows acquisition of high-quality angiography images after intravenous administration of contrast agent. We tested KEDSA for its suitability for intravenous cerebral angiography in an animal model. Adult male New Zealand rabbits were subjected to either angiography with conventional x-ray equipment or synchrotron-based intravenous KEDSA, using an iodine-based contrast agent. Angiography with conventional x-ray equipment after intra-arterial administration of contrast agent demonstrated the major intracranial vessels but no smaller branches. KEDSA was able to visualize the major intracranial vessels as well as smaller branches in both radiography mode (planar images) and tomography mode. Visualization was achieved with as little as 0.5 ml kg-1 of iodinated contrast material. We were able to obtain excellent visualization of the cerebral vasculature in an animal model using intravenous injection of contrast material, using synchrotron-based KEDSA.

Synchrotron FTIR microspectroscopic analysis of the effects of anti-inflammatory therapeutics on wound healing in laminectomized rats.

Peridural scarring, or the excessive formation of scar tissue following spinal surgery, is one of the important contributing factors that result in persistent pain and disability in many individuals who have undergone elective back surgery. Treatment with anti-inflammatory agents following surgery may reduce oxidative stress and scarring, leading to a reduction in post-operative pain. We are using a surgical rat model to test the hypothesis that post-surgical inflammation and oxidative stress following laminectomy can be reduced by systemic administration of L: -2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin. OTC is a cysteine precursor required for the synthesis of glutathione, an important antioxidant. Quercetin is a flavonoid with anti-oxidant properties, found in fruits and vegetables. Synchrotron FTIR microspectroscopy data has been collected on OTC, quercetin and saline (control)-treated post-surgery animals, sacrificed at 3 and 21 days (n = 6 per age and treatment group). This paper presents preliminary IR results, supported by immunocytochemistry, on the heterogeneous distribution of biological components present in the healing tissue. The data collected on animals sacrificed at 3 and 21 days post-surgery will be combined in the future with data from animals sacrificed 63 days after surgery (representing a third time point) to evaluate the efficacy of the different treatments. Initial statistical analysis of ED1 immunohistochemistry results indicates a decrease in the number of activated macrophages 21 days post-surgery in the OTC-treated animals compared with the saline controls.

Diffraction-enhanced imaging of the rat spine.

INTRODUCTION: Diffraction-enhanced imaging (DEI) uses monochromatic synchrotron X-rays to image tissue. This technique has been shown to produce superior bony and soft tissue characterization when compared with conventional absorption radiography. Application of this imaging modality is under investigation, and this study represents the first DEI analysis of the vertebral column. METHODS: Four male Wistar rats were studied. Spine muscle blocks were imaged in 3 of the rats after thoracic laminectomy (n = 1), after lumbar laminectomy (n = 1), and in a control condition (n = 1). The fourth rat was imaged as a whole animal control. Conventional radiography and synchrotron-supported DEI at 40 keV were performed on all specimens. We compared images side by side, using a nonvalidated subjective assessment technique. RESULTS: DEI produced superior visualization of the vertebral anatomy, compared with conventional absorption radiography for all specimens. Greater bony and soft tissue detail was noted, with improved image contrast. In addition to imaging the anatomical structures, DEI showed the polyglactin suture material used for fascial closure in the 2 animals that underwent surgery. Artifact from air bubbles was present on DEI images but not on plain radiographs. CONCLUSIONS: This represents the first use of DEI, a novel imaging modality, to image the vertebral column. It provides excellent anatomic detail with superior contrast and visualization of both bone and soft tissue when compared with conventional radiography. Future applications of this investigational technique may include analysis of spinal fusion as well as degenerative and neoplastic conditions of the spine.

The effect of glutamine on locomotor performance and skeletal muscle myosins following spinal cord injury in rats.

Following initial spinal cord injury (SCI), a cascade of pathological events, including oxidative stress, leads to secondary injury. Glutathione (GSH) plays a critical role in oxidant scavenging. Maintenance of GSH concentrations after SCI lessens secondary injury and improves recovery. Since glutamine promotes GSH synthesis, this nonessential amino acid was examined for therapeutic potential. Denervation alters the expression of myosin heavy chain (MHC) isoforms within skeletal muscles. The hypotheses of this study were that glutamine administration to SCI rats would lead to improved functional recovery and more preserved MHC phenotypes in representative locomotor muscles. Male Wistar rats were divided into four groups: healthy, sham with laminectomy, laminectomized SCI untreated, and laminectomized SCI treated with glutamine. Functional performance was measured weekly for 6 wk using Basso-Beattie-Bresnahan scale and angle board methods. MHC composition of rat soleus and extensor digitorum longus muscles was determined using SDS-PAGE. Glutamine-treated rats had significantly higher angle board scores (P < 0.001) and Basso-Beattie-Bresnahan scores (P < 0.01) than untreated SCI rats. Soleus of healthy rats contained 94% type 1 myosin isoform. Treated rats maintained 68%, which was significantly (P < 0.001) greater than 28% in untreated rats. The extensor digitorum longus of healthy rats contained 55% type 2b myosin. There was a significant (P < 0.001) decrease in this isoform following SCI, but no significant difference between treated and untreated groups. There were strong correlations between higher functional scores and more preserved MHC phenotypes. Our findings suggest glutamine improves functional recovery and helps preserve myosin profile by reducing secondary SCI, thereby maintaining more nerves.