We would like to acknowledge support from US National Institutes of Health grants R01NS057476, P50NS010828, P01NS055104, R01EB000790, K99NS067050, R01HL081273 and R01EB007279 and American Heart Association grant 0855772D.

1. Wide field of view imaging of pO2 in cortical vasculature of a rat
<ol>
 <li> Rats (250 g -350 g) are initially anesthetized with isoflurane, and the femoral artery and vein are catheterized to monitor heart rate, blood pressure, and blood gases, as well as for intravenous infusion. Body temperature is maintained at 37 &plusmn; 0.1 &deg;C. Tracheotomy is performed, and rats are ventilated with a mixture of air and oxygen.</li>
 <li> Blood samples for measuring the blood gases are taken every 30-45 min and ventilation and anesthesia are adjusted to keep the blood gases within normal physiological range.</li>
 <li> A closed cranial window on the parietal bone 4 mm x 4 mm in size is prepared for imaging. The bone and dura are removed and the cranial window is filled with 1.5% agarose and sealed with a microscope coverslip. An additional 1 mm2 burr hole on the frontal bone is used to induce CSD by intracortical microinjection of KCl (~10 &mu;l, 1 M). Extreme care should be taken to avoid any damage of the cortical vasculature. Excess heat, mechanical pressure, or brief period of hypoxia may compromise blood brain barrier and cause leakage of the dye from the vasculature into interstitial space. </li>
 <li> A mask from optically opaque material is placed around cranial window. The purpose of the mask is to absorb phosphorescence signal that is coming from the dye that leaked into the tissue from the edges of the dura matter. The dye in the interstitial space is the source of the very bright phosphorescence that can spoil the measurement. The increased brightness of the dye in interstitial space is result of build up of the dye in environment with low oxygen pressure and low quenching rate. The damage of the vasculature in experiment is therefore easily recognized by appearance of bright phosphorescence spots, in which case the experimental data is rejected.</li>
 <li> After completion of the surgery, isoflurane is discontinued and anesthesia is switched to alphachloralose (50 mg/kg intravenous bolus followed by 40 mg/(kg h) infusion). </li>
 <li> Animal is transferred on a cart with ventilator, blood pressure and temperature monitor. While breathing the room air, the animal is quickly moved to the imaging setup and reattached to the flowmeter with appropriate mixture of air and oxygen.</li>
 <li> The stereotaxic frame that holds the animal is placed under the objective. The cranial window is centered in the field of view under the objective and positioned parallel to the focal plane.</li>
 <li> The pulsed laser is turned on and set to minimal pulse energy. The optical beam is directed toward pulse energy meter and pulse energy delivered to the sample is adjusted to be not more than 10 mJ/cm2. The beam iss centered on the cranial window with oblique incidence angle of ~60 degrees and laser iss turned off.</li>
 <li> Blood gas measurements and adjustments of the ventilation and anesthesia are performed until all physiological parameters of the animal were within normal physiological range.</li>
 <li> Assuming that blood volume is approximately 7% of the body weight, an appropriate amount of the phosphorescence probe Oxyphor R3 is dissolved in 1 mL of saline to achieve 4 x 10-5 M blood concentration of the probe. The probe solution is injected via the femoral vein.</li>
 <li> A 1 M solution of potassium chloride is prepared and ~1 ul is injected with the syringe through the burr hole on the frontal bone to induce CSD.</li>
 <li> The laser is turned on and imaging is started immediately after injection of the KCl solution. Imaging of phosphorescence is performed during ~10 min.</li>
 <li> Another blood gas measurement is performed to confirm that animal physiological parameters are still within normal range.</li>
 <li> Phosphorescence lifetimes are obtained for all pixels by fitting the single exponential decay using nonlinear square fitting with statistical weighting in Matlab. Phosphorescence lifetimes are converted to the pO2 values using an empirical Stern Volmer-like relationship (vide infra).</li>
</ol>
2. High resolution two-photon pO2 imaging in cortical micro vasculature of a mouse
<ol>
 <li> Mice are anesthetized with isofluorene and femoral artery is catheterized to monitor heart rate, blood pressure, and blood gases, as well as for administration of the dye. Body temperature is maintained at 37 &plusmn; 0.1 &deg;C and animals are spontaneously breathing a mixture of air and oxygen. </li>
 <li> A cranial window is prepared according to technique originally developed by David Kleinfeld and Winfried Denk. A good care should be taken to avoid any damage of the vasculature to prevent possible leakage of the dye into interstitial space.</li>
 <li> A few blood samples for measuring the blood gases are taken during a whole preparation procedure and ventilation and anesthesia are adjusted to keep the blood gases within normal physiological range.</li>
 <li> The animal is moved to the imaging setup. A modified stereotaxic frame that holds the animal is placed under the objective. The cranial window is centered in the field of view under the Olympus 4X objective and positioned parallel to the focal plane.</li>
 <li> An image of the cranial window is taken with the digital camera through the eyepieces, and the 4X objective is replaced with the Olympus 20X objective (NA = 0.95).</li>
 <li> Blood gas measurements and adjustments of the ventilation and anesthesia are performed until all physiological parameters of the animal are within a normal physiological range.</li>
 <li> Assuming that blood volume is approximately 7% of the body weight, an appropriate amount of the phosphorescence probe PtP-C343 is dissolved in 0.2 mL of saline to achieve ~15 &mu;M blood concentration of the probe. The probe solution is injected via the femoral artery.</li>
 <li> Begin the experiment by setting the desired time intervals for dye excitation and emission collection at each pixel.</li>
 <li> Acquire a survey scan which is a slow 2-dimensional raster scan of phosphorescence intensity decay that displays the vasculature structure at the current depth. The objective is moved perpendicular to the cranial window (Z axis) and slow 2D survey scans of phosphorescence intensity are taken using the minimal laser power at 840 nm to find the microvessels in the imaging plane.</li>
 <li> At each imaging depth, based on the survey scan of phosphorescence at that depth, a set of points within the vasculature are selected, and recording of phosphorescence decays at each point is repeated for a predefined number of averaging.</li>
 <li> Set the desired amount of averaging, measurement interval, and experiment duration and start the measurement. pO2 measurements are acquired at the selected locations at the specified measurement interval for the duration of the experiment. </li>
 <li> During measurements, the software re-directs the excitation laser to the selected locations by changing the position of the galvanometer scanning mirrors. Control of the galvanometer scanners, electro optical modulator, and all other equipment are performed by custom made software in LabView.</li>
 <li> Phosphorescence lifetimes are obtained for all selected points by fitting the single exponential decay using nonlinear square fitting in Matlab. Phosphorescence lifetimes are converted to the pO2 values using an empirical Stern Volmer-like relationship (vide infra).</li>
 <li> After collecting the data at various cortical depths, inject dextran-conjugated fluorescein dye into vasculature for imaging the microvasculature structure.</li>
 <li> Obtain a stack of structural images of the vasculature by performing the two-photon imaging of FITC fluorescence using a green channel in the four-channel detector.</li>
 <li> Experiments on animals were performed in accordance with the guidelines and regulations set forth by Massachusetts General Hospital Subcommittee on Research Animal Care.</li>
</ol>
3. Representative Results:
<img src="/files/ftp_upload/1694/1694fig1.jpg" alt="Figure 1" /> Figure 1. Panel (a) on the left side of this figure displays a wide field of view image of oxygen pressure before the arrival of a CSD wave. Panel (b) on the right side shows the temporal evolution of the average oxygen pressure during CSD propagation within the region of interest marked on panel (a).
Figure 2 (avi movie): This movie shows the temporal evolution of the oxygen pressure in the whole cranial window during propagation of the CSD wave. Scale bar indicates oxygen pressure in millimeters of mercury.
<img src="/files/ftp_upload/1694/1694fig3.jpg" alt="Figure 3" /> 
Figure 3. 3D projection of the imaged vasculature stack. The shades of grey represent a volumetric vessel mask, created based on the structural image. 
Measured pO2 values are color-coded. Scale bar is 200 micrometers. Reprinted with permission from Nature Publishing Group.7

<ol>
	<li>Kleinfeld, D., Friedman, B., Lyden, P. D., Shih, A. Y., Chen, J., Xu, Z., Xu, X., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+occlusion+to+surface+and+deep+vessels+in+neocortex+via+linear+and+nonlinear+optical+absorption,+Animal+Models+of+Acute+Neurological+Injuries.">Targeted occlusion to surface and deep vessels in neocortex via linear and nonlinear optical absorption, Animal Models of Acute Neurological Injuries.</a> Contemporary Neuroscience Series. , (2007).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J Vis Exp. , (2008).</li><li>Lebedev, A. Y., Cheprakov, A. V., Sakadzic, S., Boas, D. A., Wilson, D. F., Vinogradov, S. A., A, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+Phosphorescent+Probes+for+Oxygen+Imaging+in+Biological+Systems.">Dendritic Phosphorescent Probes for Oxygen Imaging in Biological Systems.</a> Applied Materials &#38; Interfaces. , (2009).</li><li>Finikova, O. S., Lebedev, A. Y., Aprelev, A., Troxler, T., Gao, F., Garnacho, C., Muro, S., Hochstrasser, R. M., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+microscopy+by+two-photon-excited+phosphorescence.">Oxygen microscopy by two-photon-excited phosphorescence.</a> Chemphyschem. 9, 1673-1679 (2008).</li><li>Sakad&#382;i&#263;, S., Yuan, S., Dilekoz, E., Ruvinskaya, S., Vinogradov, S. A., Ayata, C., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+cerebral+partial+pressure+of+oxygen+and+blood+flow+during+functional+activation+and+cortical+spreading+depression.">Simultaneous imaging of cerebral partial pressure of oxygen and blood flow during functional activation and cortical spreading depression.</a> Appl Opt. 48, D169-D177 (2009).</li><li>Yaseen, M. A., Srinivasan, V. J., Sakadzic, S., Wu, W., Ruvinskaya, S., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+monitoring+of+oxygen+tension+in+cortical+microvessels+with+confocal+microscopy.">Optical monitoring of oxygen tension in cortical microvessels with confocal microscopy.</a> Opt Express. 17, 22341-22350 (2009).</li><li>Sakadzic, S., Roussakis, E., Yaseen, M. A., Mandeville, E. T., Srinivasan, V. J., Arai, K., Ruvinskaya, S., Devor, A., Lo, E. H., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+high-resolution+measurement+of+partial+pressure+of+oxygen+in+cerebral+vasculature+and+tissue.">Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue.</a> Nat Methods. 7, 755-759 (2010).</li></ol>

We demonstrated two applications of the pO2 measurement in cortical microvasculature based on oxygen-dependent quenching of phosphorescence. While the first method based on CCD imaging provides wide field of view monitoring of pO2, measuring partial pressure of oxygen in cortical microvasculature based on 2-photon microscopy provides capillary resolution and allows imaging in depth. Both methods provide high speed and high signal-to-noise measurements. In addition, the phosphorescence lifetime measurement of pO2 is large...

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<td>Glycopyrrolate</td>
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<td>American Regent Inc.</td>
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<td>Used to control pharyngeal, tracheal, and bronchial secretions.</td>
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<td>Used as a general inhalation anesthetic drug during surgeries and as a general anesthesia during experiments with mice.</td>
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<td>Alpha Chloralose</td>
<td>Reagent</td>
<td>Sigma</td>
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<td>Used as a general anesthesia during experiments with rats.</td>
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<td>Fluorescein isothio-cyanate&#x2013;dextran</td>
<td>Reagent</td>
<td>Sigma</td>
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cerebral blood oxygenation measurement based on oxygen-dependent quenching of phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

Watch this Scientific Journal Video about Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence at JoVE.com

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

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14.6K Views.  Massachusetts General Hospital and Harvard Medical School. We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

Video: Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Mapping Cerebral Blood Flow and Electrical Fields: Electrode Placement in Mice

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Author Spotlight: Stimulation-Based Approach to Improve Cerebral Blood Flow in Alzheimer&#039;s Model 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

Massage Manipulation in the Rat Model of Cerebral Palsy and Its Effect on Motor Dysfunction

To begin massage manipulation in the cerebral palsy rat model, first, set the desired forces for pressing and rubbing. After hypoxic ischemic encephalopathy induction, on the second day, carefully remove the young massage group rats from the cage with minimal disturbance to the maternal rats. Before massage, warm up hands by rubbing them slightly to match the skin temperature of the young rats.Position the young rat comfortably within the palm of the left hand. Gently stroke the rat's back from head to tail with the threaded surface of four fingers for one minute to quiet it. Then, rub the abdomen of the right thumb and forefinger back and forth.Massage the forelimb from the proximal to the distal end for approximately 30 seconds. Apply pressure on the Quchi, Waiguan, and Hegu points 10 times for 1 second each. Next, rub the hind legs starting with the proximal to the distal end for approximately 30 seconds.Press Yanglingquan, Zusanli, Sanyinjiao and Taichong points 10 times for 1 second each lasting approximately 5 minutes in total. Then using the right thumb, press on Zhongwan, Tianshu, Guanyuan 10 times for approximately 1 second each with the entire process lasting 1 minute. Now hold the young rat in a prone position and apply pressure using the right thumb on the Zhongwan point located on the abdomen.Keep the navel of the rat as the center and rotate the thumb 120 times per minute for about five minutes in a clockwise direction. Using the right hands, four fingers, gently stroke the rat from its head to its tail, using the thread like surface to calm it down. Once calm, place the young rat back in the cage and allow the mother to feed it After a span of 2 weeks to 40 days, administer regular rat massage therapy to all the acu points shown earlier.Next, weigh the young rats daily from day 7 to day 40. Place each young rat on the balance beam, directing it towards a black box at the opposite end. Conduct balance beam training for the subject on days 37 to 39, 3 times daily, and a concluding test on day 40.Record the time it takes for each rat to cross the balance beam and the frequency of hind limb slips. After ischemia and hypoxia modeling, the growth rate of body weight in the model group decreased significantly. Massage therapy significantly improved it suggesting that massage improves the growth and development of young rats with cerebral palsy.The balance beam test results showed that the model group took longer to cross the beam than the sham and massage groups. In contrast, the number of hind limb slips significantly decreased in the massage group compared with the model group.