Name: biochemical measurement of neonatal hypoxia
Uploaded: 2011-08-24T13:00:00
Description: Watch this Scientific Journal Video about Biochemical Measurement of Neonatal Hypoxia at JoVE.com

This work is funded by National Institutes of Health R01 NR011209-03

1. Sample Collection and Processing
<ol>
<li>Collect blood sample in a 6ml K3E EDTA K3 tube which is kept on ice.</li>
<li>Within 2 min of collection, centrifuge the sample at 4&deg;C at 1500 g for 10 min.</li>
<li>Transfer the supernatant (plasma) to a 1.5ml microcentrifuge tube.</li>
<li>Centrifuge at 4&deg;C at 18000 g for 30 min.</li>
<li>Remove the supernatant aliquots and transfer them into separate microcentrifuge tubes for purine (200&mu;l), allantoin (50&mu;l), MDA (100&mu;l), and XO (120&mu;l) analysis. Be c...

<ol>
	<li>Harkness, R. A., Whitelaw, A. G., Simmonds, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrapartum+hypoxia:+the+association+between+neurological+assessment+of+damage+and+abnormal+excretion+of+ATP+metabolites.">Intrapartum hypoxia: the association between neurological assessment of damage and abnormal excretion of ATP metabolites.</a> J Clin Pathol. 35, 999-1007 (1982).</li><li>Shalak, L., Perlman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxic-ischemic+brain+injury+in+the+term+infant-current+concepts.">Hypoxic-ischemic brain injury in the term infant-current concepts.</a> Early Hum Dev. 80, 125-141 (2004).</li><li>Webster, W. S., Abela, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+hypoxia+in+development.">The effect of hypoxia in development.</a> Birth Defects Res C Embryo Today. 81, 215-228 (2007).</li><li>Engerson, T. D., McKelvey, T. G., Rhyne, D. B., Boggio, E. B., Snyder, S. J., Jones, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+xanthine+dehydrogenase+to+oxidase+in+ischemic+rat+tissues.">Conversion of xanthine dehydrogenase to oxidase in ischemic rat tissues.</a> J Clin Invest. 79, 1564-1570 (1987).</li><li>Choi, E. Y., Stockert, A. L., Leimkuhler, S., Hille, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanism+of+action+of+xanthine+oxidase.">Studies on the mechanism of action of xanthine oxidase.</a> J Inorg Biochem. 98, 841-848 (2004).</li><li>Godber, B. L., Schwarz, G., Mendel, R. R., Lowe, D. J., Bray, R. C., Eisenthal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+human+xanthine+oxidoreductase:+the+enzyme+is+grossly+deficient+in+molybdenum+and+substantially+deficient+in+iron-sulphur+centres.">Molecular characterization of human xanthine oxidoreductase: the enzyme is grossly deficient in molybdenum and substantially deficient in iron-sulphur centres.</a> Biochem J. 388, 501-508 (2005).</li><li>Gruber, J., Tang, S. Y., Jenner, A. M., Mudway, I., Blomberg, A., Behndig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allantoin+in+human+plasma,+serum,+and+nasal-lining+fluids+as+a+biomarker+of+oxidative+stress:+avoiding+artifacts+and+establishing+real+in+vivo+concentrations.">Allantoin in human plasma, serum, and nasal-lining fluids as a biomarker of oxidative stress: avoiding artifacts and establishing real in vivo concentrations.</a> Antioxid Redox Signal. 11, 1767-1776 (2009).</li><li>Zitnanova, I., Korytar, P., Aruoma, O. I., Sustrova, M., Garaiova, I., Muchova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uric+acid+and+allantoin+levels+in+Down+syndrome:+antioxidant+and+oxidative+stress+mechanisms?.">Uric acid and allantoin levels in Down syndrome: antioxidant and oxidative stress mechanisms?.</a> Clin Chim Acta. 341, 139-146 (2004).</li><li>Siciarz, A., Weinberger, B., Witz, G., Hiatt, M., Hegyi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+thiobarbituric+acid-reacting+substances+as+potential+biomarkers+of+intrauterine+hypoxia.">Urinary thiobarbituric acid-reacting substances as potential biomarkers of intrauterine hypoxia.</a> Arch Pediatr Adolesc Med. 155, 718-722 (2001).</li><li>Buonocore, G., Perrone, S., Longini, M., Terzuoli, L., Bracci, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+hydroperoxide+and+advanced+oxidation+protein+products+in+preterm+hypoxic+babies.">Total hydroperoxide and advanced oxidation protein products in preterm hypoxic babies.</a> Pediatr Res. 47, 221-224 (2000).</li><li>Berne, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+nucleotides+in+hypoxia:+possible+role+in+regulation+of+coronary+blood+flow.">Cardiac nucleotides in hypoxia: possible role in regulation of coronary blood flow.</a> Am J Physiol. 204, 317-322 (1963).</li><li>Harkness, R. A., Lund, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+concentrations+of+hypoxanthine,+xanthine,+uridine+and+inosine:+high+concentrations+of+the+ATP+metabolite,+hypoxanthine,+after+hypoxia.">Cerebrospinal fluid concentrations of hypoxanthine, xanthine, uridine and inosine: high concentrations of the ATP metabolite, hypoxanthine, after hypoxia.</a> J Clin Pathol. 36, 1-8 (1983).</li><li>Plank, M. S., Boskovic, D. S., Sowers, L. C., Angeles, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+markers+of+neonatal+hypoxia.">Biochemical markers of neonatal hypoxia.</a> Pediatric Health. 2, 485-501 (2008).</li><li>Cighetti, G., Allevi, P., Anastasia, L., Bortone, L., Paroni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+methyl+malondialdehyde+as+an+internal+standard+for+malondialdehyde+detection:+validation+by+isotope-dilution+gas+chromatography-mass+spectrometry.">Use of methyl malondialdehyde as an internal standard for malondialdehyde detection: validation by isotope-dilution gas chromatography-mass spectrometry.</a> Clin Chem. 48, 2266-2269 (2002).</li><li>Paroni, R., Fermo, I., Cighetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+methyl+malondialdehyde+as+internal+standard+for+malondialdehyde+detection+by+capillary+electrophoresis.">Validation of methyl malondialdehyde as internal standard for malondialdehyde detection by capillary electrophoresis.</a> Anal Biochem. 307, 92-98 (2002).</li><li>Cighetti, G., Debiasi, S., Ciuffreda, P., Allevi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-ethoxyacrolein+contamination+increases+malondialdehyde+inhibition+of+milk+xanthine+oxidase+activity.">Beta-ethoxyacrolein contamination increases malondialdehyde inhibition of milk xanthine oxidase activity.</a> Free Radic Biol Med. 25, 818-825 (1998).</li><li>Cighetti, G., Debiasi, S., Paroni, R., Allevi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+and+total+malondialdehyde+assessment+in+biological+matrices+by+gas+chromatography-mass+spectrometry:+what+is+needed+for+an+accurate+detection.">Free and total malondialdehyde assessment in biological matrices by gas chromatography-mass spectrometry: what is needed for an accurate detection.</a> Anal Biochem. 266, 222-229 (1999).</li><li>Yamamoto, T., Moriwaki, Y., Takahashi, S., Tsutsumi, Z., Yamakita, J., Nasako, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+human+plasma+xanthine+oxidase+activity+by+high-performance+liquid+chromatography.">Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography.</a> J Chromatogr B Biomed Appl. 681, 395-400 (1996).</li><li>Fasman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Biochemistry and Molecular Biology. , (1988).</li><li>Chen, X. B., Calder, A. G., Prasitkusol, P., Kyle, D. J., Jayasuriya, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+15N+isotopic+enrichment+and+concentrations+of+allantoin+and+uric+acid+in+urine+by+gas+chromatography/mass+spectrometry.">Determination of 15N isotopic enrichment and concentrations of allantoin and uric acid in urine by gas chromatography/mass spectrometry.</a> J Mass Spectrom. 33, 130-137 (1998).</li></ol>

The methods described here permit the evaluation of neonatal hypoxia ischemia. This protocol combines the measurements of markers of energy (ATP) deprivation, oxidative stress, oxidative damage, and enzyme activity to gain an overall biochemical picture of the presence or even the degree of hypoxic ischemia. Despite the usefulness of this method, there are potential limitations. Firstly, it takes roughly 1-2 ml of blood to collect enough plasma to run all of the assays. This will not be a problem in adults or childre...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>6ml K3E EDTA K3 tube</td>
			<td>Fisher Scientific</td>
			<td>2204061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5702R centrifuge</td>
			<td>Fisher Scientific</td>
			<td>05413319</td>
			<td>With 13&amp;16MM adaptor</td>
		</tr>
		<tr>
			<td>1.5ml microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5599</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Aminopurine</td>
			<td>Sigma-Aldrich</td>
			<td>A3509</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Varian Cary 100 spectrophotometer</td>
			<td>Agilant Technologies</td>
			<td>0010071500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Savant SpeedVac</td>
			<td>Thermo Scientific</td>
			<td>SC210A-115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micron centrifugal filter device</td>
			<td>Fisher Scientific</td>
			<td>UFC501596</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Supelcosil LC-18-S Column</td>
			<td>Sigma-Aldrich</td>
			<td>58931</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Supelcosil LC-18-S Supelguard cartridge and holder</td>
			<td>Sigma-Aldrich</td>
			<td>59629</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Waters</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GCMS Vial</td>
			<td>Fisher Scientific</td>
			<td>03376607</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DL-Allantoin-5-13C;1-15N</td>
			<td>CDN Isotopes</td>
			<td>M-2307</td>
			<td>Lot #L340P9</td>
		</tr>
		<tr>
			<td>MTBSTFA</td>
			<td>Thermo Scientific</td>
			<td>48920</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pyridine </td>
			<td>Sigma-Aldrich</td>
			<td>270970</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5973E GC/MSD</td>
			<td>Agilent Technologies</td>
			<td>G7021A</td>
			<td>Part # for 5975E GC/MS</td>
		</tr>
		<tr>
			<td>3-Ethoxymethacrolein</td>
			<td>Sigma-Aldrich</td>
			<td>232548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benzene</td>
			<td>Sigma-Aldrich</td>
			<td>401765</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Diisopropyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>38270</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BHT</td>
			<td>Sigma-Aldrich</td>
			<td>B1378</td>
			<td>&nbsp;</td>
		</tr>
		
		<tr>
			<td>Ethanol </td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenylhydrazine</td>
			<td>Sigma-Aldrich</td>
			<td>P26252</td>
			<td>&nbsp;</td>
		</tr>
		
	</tbody>
</table>

biochemical measurement of neonatal hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

Watch this Scientific Journal Video about Biochemical Measurement of Neonatal Hypoxia at JoVE.com

Biochemical Measurement of Neonatal Hypoxia

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

biochemical-measurement-of-neonatal-hypoxia

Research

JoVE Journal

Medicine

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. Collecting site-specific biochemical markers is important to understand the relationship between levels in serum and surgical wound, determine any associations between mediator release, pain, analgesic use and other outcomes of interest, and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry. This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. We have measured wound exudate and serum mediators at the same time intervals as patient's pain scores and analgesics consumption for up to 48 hours post-cesarean delivery. Using this methodology we have been able to detect various biochemical mediators including nerve growth factor (NGF), prostaglandin E2 (PG-E2) substance P, IL-1&#x03B2;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&#x03B3;, G-CSF, GM-CSF, MCP-1 and MIP-1&#x03B2;. Studies applying this human surgical wound bioassay have found no correlations between wound and serum cytokine concentrations or their time-release profile (J Pain. 2008; 9(7):650-7).1 We also documented the utility of the technique to identify drug-mediated changes in wound cytokine content (Anesth Analg 2010; 111:1452-9).2

This article provides a detailed and visual description of a methodology for collecting and measuring biochemical inflammatory and nociceptive mediators at the surgical wound site following cesarean delivery. This human bioassay has been used to determine correlations between wound and serum cytokine concentrations and drug-mediated changes in wound cytokines, chemokines and neuropetides.

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...