Martin M. Pike

Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus

Cilia are complex organelles involved in sensory perception and fluid or cell movement. They are constructed through a highly conserved process called intraflagellar transport (IFT). Mutations in IFT genes, such as Tg737, result in severe developmental defects and disease. In the case of the Tg737orpk mutants, these pathological alterations include cystic kidney disease, biliary and pancreatic duct abnormalities, skeletal patterning defects, and hydrocephalus. Here, we explore the connection between cilia dysfunction and the development of hydrocephalus by using the Tg737orpk mutants. Our analysis indicates that cilia on cells of the brain ventricles of Tg737orpk mutant mice are severely malformed. On the ependymal cells, these defects lead to disorganized beating and impaired cerebrospinal fluid (CSF) movement. However, the loss of the cilia beat and CSF flow is not the initiating factor, as the pathology is present prior to the development of motile cilia on these cells and CSF flow is not impaired at early stages of the disease. Rather, our results suggest that loss of cilia leads to altered function of the choroid plexus epithelum, as evidenced by elevated intracellular cAMP levels and increased chloride concentration in the CSF. These data suggest that cilia function is necessary for regulating ion transport and CSF production, as well as for CSF flow through the ventricles.

Quantification of [Ca2+]i in perfused hearts. Critical evaluation of the 5F-BAPTA and nuclear magnetic resonance method as applied to the study of ischemia and reperfusion.

Calcium has been implicated as a mediator of cell injury in ischemia and reperfusion, but direct measurements of Ca2+ are required to refine this idea. We used nuclear magnetic resonance spectroscopy and the Ca2+ indicator 5F-BAPTA to measure [Ca2+]i in perfused ferret hearts. Several lines of evidence are presented to show that loading with the acetoxymethyl ester of 5F-BAPTA is not significantly complicated by accumulation of partially de-esterified metabolites, compartmentalization into mitochondria, or disproportionate uptake into endothelial cells. During 20 minutes of total global ischemia at 30 degrees C, time-averaged [Ca2+]i increased significantly, reaching peak values roughly three times control at 15-20 minutes. Reperfusion resulted in a persistent elevation of [Ca2+]i during the first 5 minutes, but not afterward. Although the nonlinear response of 5F-BAPTA to [Ca2+] leads to underestimation of the true time-averaged [Ca2+]i, the measured alterations of intracellular Ca2+ homeostasis during ischemia are large compared with the likely errors in quantification. Phosphorus nuclear magnetic resonance spectroscopy of 5F-BAPTA-loaded hearts reveals changes during ischemia similar to those recorded previously in hearts not containing a Ca2+ indicator. Developed pressure recovers to only 50% of control values during reflow, indicating that the presence of 5F-BAPTA in the cytosol does not protect against stunning, at least when the extracellular calcium concentration has been raised to 8 mM. We conclude that 5F-BAPTA provides useful measurements that reveal that time-averaged [Ca2+]i rises during ischemia and returns to control levels soon after reperfusion.

Dynamic MRI using iron oxide nanoparticles to assess early vascular effects of antiangiogenic versus corticosteroid treatment in a glioma model

The vascular effects of antiangiogenic treatment may pose problems for evaluating brain tumor response based on contrast-enhanced magnetic resonance imaging (MRI). We used serial dynamic contrast-enhanced MRI at 12 T to assess vascular responses to antiangiogenic versus steroid therapy. Athymic rats with intracerebral U87MG human glioma (n = 17) underwent susceptibility-weighted perfusion MRI with ferumoxytol, a solely intravascular ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle, followed by T1-weighted dynamic gadodiamide-enhanced MRI to measure vascular permeability. Rats were imaged before and after 24, 48, and 72 h of treatment with the antiangiogenic agent bevacizumab or the corticosteroid dexamethasone. Contrast agent extravasation was seen rapidly after gadodiamide, but not with ferumoxytol administration. Bevacizumab significantly decreased the blood volume and decreased permeability in tumors as determined by increased time-to-peak enhancement. A single dose of 45 mg/kg bevacizumab resulted in changes analogous to dexamethasone given in an extremely high dose (12 mg/kg per day), and was significantly more effective than dexamethasone at 2 mg/kg per day. We conclude that dynamic perfusion MRI measurements with ferumoxytol USPIO to assess cerebral blood volume, along with dynamic gadodiamide-enhanced MR to assess vascular permeability, hold promise in more accurately detecting therapeutic responses to antiangiogenic therapy.

A new model of cortical stroke in the rhesus macaque.

Primate models are essential tools for translational research in stroke but are reportedly inconsistent in their ability to produce cortical infarcts of reproducible size. Here, we report a new stroke model using a transorbital, reversible, two-vessel occlusion approach in male rhesus macaques that produces consistent and reproducible cortical infarcts. The right middle cerebral artery (distal to the orbitofrontal branch) and both anterior cerebral arteries were occluded with vascular clips. Bilateral occlusion of the anterior cerebral artery was critical for reducing collateral flow to the ipsilateral cortex. Reversible ischemia was induced for 45, 60, or 90 mins (n = 2/timepoint) and infarct volume and neurologic outcome were evaluated. The infarcts were located predominantly in the cortex and increased in size with extended duration of ischemia determined by T2-weighted magnetic resonance imaging. Infarct volume measured by 2,3,5-triphenyl tetrazolium chloride and cresyl violet staining corroborated magnetic resonance imaging results. Neurologic deficit scores worsened gradually with longer occlusion times. A subset of animals (n = 5) underwent 60 mins of ischemia resulting in consistent infarct volumes primarily located to the cortex that correlated well with neurologic deficit scores. This approach offers promise for evaluating therapeutic interventions in stroke.

23Na and 31P Nuclear Magnetic Resonance Studies of Ischemia-Induced Ventricular Fibrillation : Alterations of Intracellular Na+ and Cellular Energy

To clarify the role of Na+i, pHi, and high-energy phosphate (HEP) levels in the initiation and maintenance of ischemia-induced ventricular fibrillation (VF), interleaved 23Na and 31P nuclear magnetic resonance spectra were collected on perfused rat hearts during low-flow ischemia (51 minutes, 1.2 mL/g wet wt). When untreated, 50% of the hearts from normal (sham) rats and 89% of the hypertrophied hearts from aorticbanded (band) rats (P < .01 versus sham) exhibited VF. Phosphocreatine content was significantly higher in sham than band hearts during control perfusion (53.3 +/- 1.6 versus 39.8 +/- 2.0 mumol/g dry wt). Before VF at 20 minutes of ischemia, Na+i accumulation was greater in hearts that eventually developed VF than in hearts that did not develop VF for both band and sham groups (144% versus 128% of control in sham; P < .005) and was the strongest metabolic predictor of VF; ATP depletion was also greater for VF hearts in the sham group. Infusion of the Na(+)-H+ exchange inhibitor 5-(N,N-hexamethylene)-amiloride prevented VF in sham and band hearts; reduced Na+i accumulation but similar HEP depletion were observed compared with VF hearts before the onset of VF. Rapid changes in Na+i, pHi, and HEP began with VF, resulting in intracellular Na+i overload (approximately 300% of control) and increased HEP depletion. A delayed postischemic functional recovery occurred in VF hearts, which correlated temporally with the recovery of Na+i. In conclusion, alterations in Na+i were associated with spontaneous VF transitions, consistent with involvement of excess Na+i accumulation in VF initiation and maintenance and with previously reported alterations in Ca2+i with VF.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution longitudinal assessment of flow and permeability in mouse glioma vasculature: Sequential small molecule and SPIO dynamic contrast agent MRI.

The poor prognosis associated with malignant glioma is largely attributable to its invasiveness and robust angiogenesis. Angiogenesis involves host–tumor interaction and requires in vivo evaluation. Despite their versatility, few studies have used mouse glioma models with perfusion MRI approaches, and generally lack longitudinal study design. Using a micro‐MRI system (8.5 Tesla), a novel dual bolus‐tracking perfusion MRI strategy was implemented. Using the small molecule contrast agent Magnevist, dynamic contrast enhanced MRI was implemented in the intracranial 4C8 mouse glioma model to determine Ktrans and ve, indices of tumor vascular permeability and cellularity, respectively. Dynamic susceptibility contrast MRI was subsequently implemented to assess both cerebral blood flow and volume, using the macromolecular superparamagnetic iron oxide, Feridex, which circumvented tumor bolus susceptibility curve distortions from first‐pass extravasation. The high‐resolution parametric maps obtained over 4 weeks, indicated a progression of tumor vascularization, permeability, and decreased cellularity with tumor growth. In conclusion, a comprehensive array of key parameters were reliably quantified in a longitudinal mouse glioma study. The syngeneic 4C8 intracerebral mouse tumor model has excellent characteristics for studies of glioma angiogenesis. This approach provides a useful platform for noninvasive and highly diagnostic longitudinal investigations of anti‐angiogenesis strategies in a relevant orthotopic animal model. Magn Reson Med 61:615–625, 2009.

Nuclear Magnetic Resonance Studies of Cationic and Energetic Alterations With Oxidant Stress in the Perfused Heart: Modulation With Pyruvate and Lactate

The postischemic generation of oxygen-derived free radicals may contribute to myocardial reperfusion injury by affecting sarcolemmal ion transport. Recent evidence indicates that exposure to reactive oxygen intermediates induces rapid increases in myocardial cytosolic free Ca2+ (Ca2+i). The mechanism is undetermined but may involve disturbances in Na+ homeostasis. We tested this hypothesis by interleaving 23Na and 31P nuclear magnetic resonance (NMR) measurements of Na+i and high-energy phosphates in glucose-perfused rat hearts exposed to hydroxyl radicals generated from H2O2 and Fe3+. In separate experiments, K+i and Ca2+i were measured with 39K and 19F NMR, respectively. The hearts rapidly exhibited contracture. Threefold Na+i increases and substantial K+i depletion were observed. Glycolytic inhibition was indicated by rapid sugar phosphate accumulation and cellular energy depletion. Notably, however, severe functional and energetic deterioration and substantial elevation of Ca2+i occurred before substantial Na+i accumulation or K+i depletion was observed. Further experiments investigated the ability of pyruvate to scavenge H2O2 and to protect the myocardium from oxidant stress. Pyruvate (1 or 2.5 mmol/L) dramatically attenuated functional and energetic alterations and alterations in Na+i and K+i, whereas acetate (2.5 mmol/L) offered no protection. Unlike pyruvate, lactate (5 mmol/L) has little or no capacity to scavenge H2O2 but has similar protective effects. In conclusion, pyruvate effectively protects against H2O2/Fe3+, largely by direct H2O2 scavenging. Protection with lactate may involve intracellular pyruvate augmentation. Without exogenous pyruvate or lactate, myocardial Na+ homeostasis can be substantially altered by oxidant stress, possibly via cellular energy depletion. Excess Na+i accumulation may, in turn, hasten metabolic and functional deterioration, but a causal link with the initial alterations in function or Ca2+i was not supported.

High fat diet-induced diabetes in mice exacerbates cognitive deficit due to chronic hypoperfusion:

Diabetes causes endothelial dysfunction and increases the risk of vascular cognitive impairment. However, it is unknown whether diabetes causes cognitive impairment due to reductions in cerebral blood flow or through independent effects on neuronal function and cognition. We addressed this using right unilateral common carotid artery occlusion to model vascular cognitive impairment and long-term high-fat diet to model type 2 diabetes in mice. Cognition was assessed using novel object recognition task, Morris water maze, and contextual and cued fear conditioning. Cerebral blood flow was assessed using arterial spin labeling magnetic resonance imaging. Vascular cognitive impairment mice showed cognitive deficit in the novel object recognition task, decreased cerebral blood flow in the right hemisphere, and increased glial activation in white matter and hippocampus. Mice fed a high-fat diet displayed deficits in the novel object recognition task, Morris water maze and fear conditioning tasks and neuronal loss, but no impairments in cerebral blood flow. Compared to vascular cognitive impairment mice fed a low fat diet, vascular cognitive impairment mice fed a high-fat diet exhibited reduced cued fear memory, increased deficit in the Morris water maze, neuronal loss, glial activation, and global decrease in cerebral blood flow. We conclude that high-fat diet and chronic hypoperfusion impair cognitive function by different mechanisms, although they share commons features, and that high-fat diet exacerbates vascular cognitive impairment pathology.

1H-NMR spectroscopy can accurately quantitate the lipolysis and oxidation of cardiac triacylglycerols

Triacylglycerol metabolism in isolated, perfused hearts from rats fed a diet containing 20% rapeseed oil (RSO) was studied using 1H-NMR spectroscopy. RSO-induced elevation in cardiac triacylglycerols is associated with an increase in the peak area of fatty acid 1H-NMR resonances. The ratio of methyl, gamma-methylene or methylene protons adjacent to a carbon-carbon double bond to the number of methylene protons in these hearts measured by 1H-NMR spectroscopy gives values similar to those derived from previously reported chemical analyses. In addition, the triacylglycerol content of these hearts determined by chemical analysis directly correlates with their content of 1H-NMR visible fatty acid resonances. This quantitative relationship allows the real-time measurement of the rates of cardiac triacylglycerol lipolysis using 1H-NMR spectroscopy. Rates of triacylglycerol lipolysis measured using 1H-NMR spectroscopy are similar to those previously measured by chemical methods. Triacylglycerol lipolysis measured using 1H-NMR spectroscopy occurs at a significantly faster rate in hearts perfused in the presence or absence of glucose when compared to hearts perfused with glucose and acetate or medium-chain fatty acids. Finally, the rate of triacylglycerol lipolysis in glucose perfused hearts is linearly related to work output. These results demonstrate that 1H-NMR spectroscopy can accurately quantitate triacylglycerol content and metabolism in the rapeseed oil-fed rat model. 1H-NMR spectroscopic or imaging techniques may be useful in the real-time evaluation of cardiac triacylglycerol content and metabolism.

Soluble Epoxide Hydrolase in Hydrocephalus, Cerebral Edema, and Vascular Inflammation After Subarachnoid Hemorrhage

Background and Purpose— Acute communicating hydrocephalus and cerebral edema are common and serious complications of subarachnoid hemorrhage (SAH), whose causes are poorly understood. Using a mouse model of SAH, we determined whether soluble epoxide hydrolase (sEH) gene deletion protects against SAH-induced hydrocephalus and edema by increasing levels of vasoprotective eicosanoids and suppressing vascular inflammation. Methods— SAH was induced via endovascular puncture in wild-type and sEH knockout mice. Hydrocephalus and tissue edema were assessed by T2-weighted magnetic resonance imaging. Endothelial activation was assessed in vivo using T2*-weighted magnetic resonance imaging after intravenous administration of iron oxide particles linked to anti–vascular cell adhesion molecule-1 antibody 24 hours after SAH. Behavioral outcome was assessed at 96 hours after SAH with the open field and accelerated rotarod tests. Results— SAH induced an acute sustained communicating hydrocephalus within 6 hours of endovascular puncture in both wild-type and sEH knockout mice. This was followed by tissue edema, which peaked at 24 hours after SAH and was limited to white matter fiber tracts. sEH knockout mice had reduced edema, less vascular cell adhesion molecule-1 uptake, and improved outcome compared with wild-type mice. Conclusions— Genetic deletion of sEH reduces vascular inflammation and edema and improves outcome after SAH. sEH inhibition may serve as a novel therapy for SAH.