Nanoscale mapping of refractive index by using scattering-type scanning near-field optical microscopy.

We present a novel method for nanoscale reconstruction of complex refractive index by using scattering-type Scanning Near-field Optical Microscopy (s-SNOM). Our method relies on correlating s-SNOM experimental image data with computational data obtained through simulation of the classical oscillating point-dipole model. This results in assigning a certain dielectric function for every pixel of the s-SNOM images, which further results in nanoscale mapping of the refractive index. This method is employed on human erythrocytes to demonstrate the approach in a biologically relevant manner. The presented results advance the current knowledge on the capabilities of s-SNOM to extract quantitative information with nanoscale resolution from optical data sets with biological application.

Multi-modal assessment of neurovascular coupling during cerebral ischaemia and reperfusion using remote middle cerebral artery occlusion.

Hyperacute changes in cerebral blood flow during cerebral ischaemia and reperfusion are important determinants of injury. Cerebral blood flow is regulated by neurovascular coupling, and disruption of neurovascular coupling contributes to brain plasticity and repair problems. However, it is unknown how neurovascular coupling is affected hyperacutely during cerebral ischaemia and reperfusion. We have developed a remote middle cerebral artery occlusion model in the rat, which enables multi-modal assessment of neurovascular coupling immediately prior to, during and immediately following reperfusion. Male Wistar rats were subjected to remote middle cerebral artery occlusion, where a long filament was advanced intraluminally through a guide cannula in the common carotid artery. Transcallosal stimulation evoked increases in blood flow, tissue oxygenation and neuronal activity, which were diminished by middle cerebral artery occlusion and partially restored during reperfusion. These evoked responses were not affected by administration of the thrombolytic alteplase at clinically used doses. Evoked cerebral blood flow responses were fully restored at 24 h post-middle cerebral artery occlusion indicating that neurovascular dysfunction was not sustained. These data show for the first time that the rat remote middle cerebral artery occlusion model coupled with transcallosal stimulation provides a novel method for continuous assessment of hyperacute neurovascular coupling changes during ischaemia and reperfusion, and offers unique insight into hyperacute ischaemic pathophysiology.

Diabetic Phenotype in the Small Intestine of Zucker Diabetic Fatty Rats.

BACKGROUND/AIMS: In contrast to streptozotocin (STZ)-induced rodent models of diabetes, there are no thorough characterizations of the intestinal phenotype and the underlying changes in the global gene-expression of genetic models of diabetes, such as the Zucker diabetic fatty (ZDF) rat. The aim of the present study was to characterize the intestine in the ZDF rat. METHODS: The intestine of ZDF rats and lean controls was examined macroscopically and histologically, and ribonucleic acid sequencing (RNAseq) was performed in samples of jejunal mucosa. RESULTS: We observed an increased mass and length of the small and large intestines in ZDF rats. RNAseq showed an increased expression of Pdk2 and Pdk4, which are involved in the regulation of glucose and fatty acid metabolism, and increased expression of genes involved in gluconeogenesis and peroxisomal beta-oxidation in jejunal mucosa. CONCLUSION: Intestinal enlargement in ZDF rats is likely driven by increased food intake, since (i) it also occurs in obese and normoglycemic Zucker fatty rats, and (ii) insulin treatment of STZ-induced diabetic rats reduced the food intake and mass of the small intestine. Results from RNAseq indicate that small intestinal epithelial cells in ZDF rats have developed insulin resistance, and support that a normal physiological effect of insulin in the enterocytes is the regulation of glucose metabolism.

The arcuate nucleus mediates GLP-1 receptor agonist liraglutide-dependent weight loss.

Liraglutide is a glucagon-like peptide-1 (GLP-1) analog marketed for the treatment of type 2 diabetes. Besides lowering blood glucose, liraglutide also reduces body weight. It is not fully understood how liraglutide induces weight loss or to what degree liraglutide acts directly in the brain. Here, we determined that liraglutide does not activate GLP-1-producing neurons in the hindbrain, and liraglutide-dependent body weight reduction in rats was independent of GLP-1 receptors (GLP-1Rs) in the vagus nerve, area postrema, and paraventricular nucleus. Peripheral injection of fluorescently labeled liraglutide in mice revealed the presence of the drug in the circumventricular organs. Moreover, labeled liraglutide bound neurons within the arcuate nucleus (ARC) and other discrete sites in the hypothalamus. GLP-1R was necessary for liraglutide uptake in the brain, as liraglutide binding was not seen in Glp1r(-/-) mice. In the ARC, liraglutide was internalized in neurons expressing proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Electrophysiological measurements of murine brain slices revealed that GLP-1 directly stimulates POMC/CART neurons and indirectly inhibits neurotransmission in neurons expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP) via GABA-dependent signaling. Collectively, our findings indicate that the GLP-1R on POMC/CART-expressing ARC neurons likely mediates liraglutide-induced weight loss.

Increased 20-HETE synthesis explains reduced cerebral blood flow but not impaired neurovascular coupling after cortical spreading depression in rat cerebral cortex.

Cortical spreading depression (CSD) is associated with release of arachidonic acid, impaired neurovascular coupling, and reduced cerebral blood flow (CBF), caused by cortical vasoconstriction. We tested the hypothesis that the released arachidonic acid is metabolized by the cytochrome P450 enzyme to produce the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE), and that this mechanism explains cortical vasoconstriction and vascular dysfunction after CSD. CSD was induced in the frontal cortex of rats and the cortical electrical activity and local field potentials recorded by glass microelectrodes, CBF by laser Doppler flowmetry, and tissue oxygen tension (tpO(2)) using polarographic microelectrodes. 20-HETE synthesis was measured in parallel experiments in cortical brain slices exposed to CSD. We used the specific inhibitor HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) to block 20-HETE synthesis. CSD increased 20-HETE synthesis in brain slices for 120 min, and the time course of the increase in 20-HETE paralleled the reduction in CBF after CSD in vivo. HET0016 blocked the CSD-induced increase in 20-HETE synthesis and ameliorated the persistent reduction in CBF, but not the impaired neurovascular coupling after CSD. These findings suggest that CSD-induced increments in 20-HETE cause the reduction in CBF after CSD and that the attenuation of stimulation-induced CBF responses after CSD has a different mechanism. We suggest that blockade of 20-HETE synthesis may be clinically relevant to ameliorate reduced CBF in patients with migraine and acute brain cortex injuries.

Activity-dependent increases in local oxygen consumption correlate with postsynaptic currents in the mouse cerebellum in vivo.

Evoked neural activity correlates strongly with rises in cerebral metabolic rate of oxygen (CMRO(2)) and cerebral blood flow (CBF). Activity-dependent rises in CMRO(2) fluctuate with ATP turnover due to ion pumping. In vitro studies suggest that increases in cytosolic Ca(2+) stimulate oxidative metabolism via mitochondrial signaling, but whether this also occurs in the intact brain is unknown. Here we applied a pharmacological approach to dissect the effects of ionic currents and cytosolic Ca(2+) rises of neuronal origin on activity-dependent rises in CMRO(2). We used two-photon microscopy and current source density analysis to study real-time Ca(2+) dynamics and transmembrane ionic currents in relation to CMRO(2) in the mouse cerebellar cortex in vivo. We report a direct correlation between CMRO(2) and summed (i.e., the sum of excitatory, negative currents during the whole stimulation period) field EPSCs (∑fEPSCs) in Purkinje cells (PCs) in response to stimulation of the climbing fiber (CF) pathway. Blocking stimulus-evoked rises in cytosolic Ca(2+) in PCs with the P/Q-type channel blocker ω-agatoxin-IVA (ω-AGA), or the GABA(A) receptor agonist muscimol, did not lead to a time-locked reduction in CMRO(2), and excitatory synaptic or action potential currents. During stimulation, neither ω-AGA or (µ-oxo)-bis-(trans-formatotetramine-ruthenium) (Ru360), a mitochondrial Ca(2+) uniporter inhibitor, affected the ratio of CMRO(2) to fEPSCs or evoked local field potentials. However, baseline CBF and CMRO(2) decreased gradually with Ru360. Our data suggest that in vivo activity-dependent rises in CMRO(2) are correlated with synaptic currents and postsynaptic spiking in PCs. Our study did not reveal a unique role of neuronal cytosolic Ca(2+) signals in controlling CMRO(2) increases during CF stimulation.

Cyclosporine A, FK506, and NIM811 ameliorate prolonged CBF reduction and impaired neurovascular coupling after cortical spreading depression.

Cortical spreading depression (CSD) is associated with mitochondrial depolarization, increasing intracellular Ca(2+), and the release of free fatty acids, which favor opening of the mitochondrial permeability transition pore (mPTP) and activation of calcineurin (CaN). Here, we test the hypothesis that cyclosporine A (CsA), which blocks both mPTP and CaN, ameliorates the persistent reduction of cerebral blood flow (CBF), impaired vascular reactivity, and a persistent rise in the cerebral metabolic rate of oxygen (CMRO(2)) following CSD. In addition to CsA, we used the specific mPTP blocker NIM811 and the specific CaN blocker FK506. Cortical spreading depression was induced in rat frontal cortex. Electrocortical activity was recorded by glass microelectrodes, CBF by laser Doppler flowmetry, and tissue oxygen tension with polarographic microelectrodes. Electrocortical activity, basal CBF, CMRO(2), and neurovascular and neurometabolic coupling were unaffected by all three drugs under control conditions. NIM811 augmented the rise in CBF observed during CSD. Cyclosporine A and FK506 ameliorated the persistent decrease in CBF after CSD. All three drugs prevented disruption of neurovascular coupling after CSD; the rise in CMRO(2) was unchanged. Our data suggest that blockade of mPTP formation and CaN activation may prevent persistent CBF reduction and vascular dysfunction after CSD.

Lack of potassium channel induces proliferation and survival causing increased neurogenesis and two-fold hippocampus enlargement.

The megencephaly mice show dramatic progressive increase in brain size and seizures. The overgrowth affects primarily the hippocampus and ventral cortex. The phenotype originates from a mutation in the Shaker-like voltage-gated potassium channel Kv1.1 brain, which results in a malfunctioning protein. A key question in elucidating the mechanism behind the unique brain overgrowth is whether it is caused by an increase in cell number. By applying stereological techniques, we found that the number of both neurons and astrocytes, as well as structure volume, was increased approximately two-fold within dentate gyrus (DG), CA2/3, and hilus of 12-week-old mceph/mceph versus wild type mice. In CA1, there was a tendency toward an increase in volume and in number of astrocytes. The volume estimates in newborn and p14 mice suggest that the overgrowth in mceph/mceph hippocampus starts between birth and the second week of life. To investigate the hyperplasia, cell proliferation was studied within the subgranular zone of the DG using BrdU and Ki67. There was a three-fold increase in proliferation in mceph/mceph mice compared to wild type mice at an age before onset of epileptic symptoms (3 weeks), and these new mceph/mceph neurons showed increased migration and had a 6-week survival rate as the new neurons in wild type DG. Also when seizures were frequent in mceph/mceph (9 weeks old), the proliferation rate was three-fold higher than in wild type. The number of TUNEL-positive cells in hippocampus was lower in mceph/mceph supporting additional overgrowth mechanism than induced by seizures. In conclusion, lack of a functional Kv1.1 ion channel subunit in the mceph/mceph mice causes a unique neuronal hyperplasia in distinct hippocampal regions and consequently hippocampal enlargement from 2 to 3 weeks of age. This phenotype is a result, at least in DG, from increased proliferation, neurogenesis, and enhanced general hippocampal cell survival.

Acute cognitive impairment after lateral fluid percussion brain injury recovers by 1 month: evaluation by conditioned fear response.

Conditioned fear associates a contextual environment and cue stimulus to a foot shock in a single training trial, where fear expressed to the trained context or cue indicates cognitive performance. Lesion, aspiration or inactivation of the hippocampus and amygdala impair conditioned fear to the trained context and cue, respectively. Moreover, only bilateral experimental manipulations, in contrast to unilateral, abolish cognitive performance. In a model of unilateral brain injury, we sought to test whether a single lateral fluid percussion brain injury impairs cognitive performance in conditioned fear. Brain-injured mice were evaluated for anterograde cognitive deficits, with the hypothesis that acute injury-induced impairments improve over time. Male C57BL/6J mice were brain-injured, trained at 5 or 27 days post-injury, and tested 48h later for recall of the association between the conditioned stimuli (trained context or cue) and the unconditioned stimulus (foot shock) by quantifying fear-associated freezing behavior. A significant anterograde hippocampal-dependent cognitive deficit was observed at 7 days in brain-injured compared to sham. Cued fear conditioning could not detect amygdala-dependent cognitive deficits after injury and stereological estimation of amygdala neuron number corroborated this finding. The absence of injury-related freezing in a novel context substantiated injury-induced hippocampal-dependent cognitive dysfunction, rather than generalized fear. Variations in the training and testing paradigms demonstrated a cognitive deficit in consolidation, rather than acquisition or recall. By 1-month post-injury, cognitive function recovered in brain-injured mice. Hence, the acute injury-induced cognitive impairment may persist while transient pathophysiological sequelae are underway, and improve as global dysfunction subsides.

Inbred mouse strains as a tool to analyze hippocampal neuronal loss after brain injury: a stereological study.

Traumatic brain injury (TBI) damages the hippocampus both in experimental animal models and in humans. In particular, the mechanical injury in combination with the genetic susceptibility to injury may result in neuronal loss from the hippocampus. This report explores the time-course of neuronal loss in the four primary subregions of the mouse hippocampus after a lateral fluid percussion injury (FPI) to the brain, and how subtle genetic differences between C57BL/6J and C57BL/10J mouse strains influence the extent and time course of neuronal loss. Using design-based stereological procedures, our results indicate negligible neuronal loss ipsilateral to the injury at 2 days postinjury in C57BL/6J mice, whereas a significant number (30-40%) of neurons are lost across all subregions of the hippocampus (dentate, hilus, area CA3, and area CA1) by 1 week, which does not appear to progress at 1 month, compared to sham. Additionally, neuronal counts after lateral FPI in a genetically similar, yet kainic acid-sensitive, mouse strain (C57BL/10J) showed no statistically significant differences in neuron number compared to the C57BL/6J strain in response to brain injury. Hippocampal neuronal loss after lateral FPI and its consequent circuit disruption may depend more on factors related to the mechanics and secondary consequences of the injury, as opposed to subtle genetic variations between inbred mouse strains. The loss of neurons appears to be restricted to the first week post-injury, and the remaining neurons may serve as a substrate for recovery.

Response of the contralateral hippocampus to lateral fluid percussion brain injury.

Traumatic brain injury is a leading cause of death and disability in the United States. Pathological examinations of humans and animal models after brain injury demonstrate hippocampal neuronal damage, which may contribute to cognitive impairments. Data from our laboratories have shown that, at 1 week after brain injury, mice possess significantly fewer neurons in all ipsilateral hippocampal subregions and a cognitive impairment. Since cognitive function is distributed across both cerebral hemispheres, the present paper explores the morphological and physiological response of the contralateral hippocampus to lateral brain injury. We analyzed the contralateral hippocampus using design-based stereology, Fluoro-Jade (FJ) histochemistry, and extracellular field recordings in mice at 7 and 30 days after lateral fluid percussion injury (FPI). At 7 days, all contralateral hippocampal subregions possess significantly fewer healthy neurons compared to sham-injured animals and demonstrate FJ-positive neuronal damage, but not at 30 days. Both the ipsilateral and contralateral dentate gyri demonstrate significantly increased excitability at 7 days post-injury, but only ipsilateral dentate gyrus hyperexcitability persists at 30 days compared to sham. In the contralateral hippocampus, the transient decrease in the number of healthy neurons, concomitant with FJ damage, and electrophysiological alterations establish a stunned period of cellular and circuit dysfunction. The return of healthy neuron number, absence of FJ damage, and sham level of excitability in the contralateral hippocampus suggest recovery of structure and function by 30 days after injury. The cognitive recovery observed after human traumatic brain injury may stem from a differential injury exposure and time course of recovery between homologous regions of the two hemispheres.

Neuronal and glial cell number in the hippocampus after experimental traumatic brain injury: analysis by stereological estimation.

Fluid percussion (FP) brain injury causes spatial memory dysfunction in rats regardless of injury location (midline vs. lateral). Standard histological analysis of the injured brains shows hippocampal neuronal loss after lateral, but not midline FP injury. We have used the optical volume fractionator (OVF) stereological procedure to quantify neuronal loss and glial proliferation within specific subregions of the hippocampus after midline or lateral FP injury. The OVF method is a design-based cell counting procedure, which combines cellular numerical density estimates (from the optical disector) with volume estimates (generated by point counting and the fractionator stereology method) to produce an estimate of the absolute cell number. Fifteen adult male Sprague-Dawley rats were randomly divided into 3 groups (n = 5/group): midline injury, lateral injury and naive. A single fluid percussion pulse was delivered to anesthetized rats in the injured groups. At 14 days post-injury, strict morphological criteria enabled the estimation of neurons, astrocytes, oligodendrocytes, and microglia in defined hippocampal subregions. The results confirm that hippocampal neurons are selectively vulnerable to brain injury, particularly observed as a significant loss in the hilus following both types of injury and in area CA3 after lateral injury. In contrast, the number of astrocytes and oligodendrocytes remains unaffected by brain injury, regardless of subregion. However, the significant increase in microglia number (bilaterally after midline and ipsilateral following lateral injury) suggests that underlying cellular processes continue weeks following injury. The implications of the observed cell population changes are discussed in relation to the reported cognitive deficits associated with both lateral and midline FP brain injury.