Special thanks to all volunteers who participated in the original study. The work of L. Eichhorn was supported through a scholarship of the Else-Kr&#246;ner-Fresenius Foundation. The authors would like to thank Springer, Part of Springer Science+Business Media, for copyright clearance (License Number 3894660871180) and the kind permission of reusing previously published data.

Ethics statement 
All procedures performed in studies involving human participants were in accordance with the ethical standards of the 1964 Helsinki declaration and its later amendments. The design of this study was approved by the local ethics committee of the University Hospital of Bonn, Germany.
NOTE: Ensure that subjects are in good and healthy condition, free of any anti-hypertensive medicine and at least 24 hours free of catecholamine inducing agents like caffeine or equal substances.
1. Preparation of the Test Subject
<ol>
	<li>Clean the skin of the forehead with 70% alcohol to degrease the skin prior to NIRS electrode positioning.</li>
	<li>Place the NIRS electrode on the right forehead above the eyebrow and to the right of the midsagittal sulcus (locus frontopolar 2) to measure cerebral (=central) tissue oxygenation.</li>
	<li>Evaluate the stability of the signal. The rSO2-signal should be constant (&#177; 3%) for at least 5 min.</li>
	<li>For measuring peripheral tissue oxygenation with NIRS (NIRStissue-electrode), place one electrode above the middle of the musculus quadriceps femoris (alternatively on the forearm). Do not place the electrode above a venous plexus or an artery.</li>
	<li>Place ECG-electrodes on the hair free chest. The ECG leads are marked with different letters. Place &#34;R&#34; on the sternocostal head of pectoralis major right, &#34;L&#34; on the sternocostal head of pectoralis major left, &#34;C&#34; on the fifth intercostal space middle of the medioclavicular line, &#34;F&#34; on the left lower rib edge, &#34;N&#34; on the right lower rib edge.</li>
	<li>Measure peripheral pulse oximetry (SpO2) on a fingertip on the same extremity and side where the NIRStissue-electrode is placed.</li>
	<li>Measure noninvasive blood pressure (NIBP) by using a blood pressure cuff. Use the contralateral extremity that allows peripheral pulse oximetry to be measured. In order to get a high time-resolution in blood pressure results, choose a one-minute interval for measuring. Choose NIBP by touching the screen and selecting &#34;settings&#34;.</li>
	<li>At least 20 min before the apnea, establish an intravenous line into the medial cubital vein of the right or left arm to draw blood samples at individual time points during and after apnea.
	<ol>
		<li>Clean the skin with 70% alcohol.</li>
		<li>Use a tourniquet to help the veins become more prominent.</li>
		<li>Use skin-disinfection to avoid infections and insert the needle through the skin.</li>
		<li>Reduce the insertion angle after blood flashback at the catheter hub. Push the catheter into the vein.</li>
		<li>Remove the needle and flush catheter with sterile saline (NaCl 0.9%).</li>
	</ol>
	</li>
</ol>
2. Data Collection
<ol>
	<li>Calibrate the internal clock of all monitors in order to synchronize measurements for later processing.
	<ol>
		<li>Click the bottom-right clock icon on the desktop, and tap &#34;change date and time settings&#34; in the pop-up window.</li>
		<li>Press the Settings menu button on the NIRS devise and change date and time via the menu.</li>
	</ol>
	</li>
	<li>To store physiological data for offline analysis, insert the monitor device into the docking station and connect it to the computer via the network cable. Ensure that the IP address and subnet mask of the docking station is correct in the network settings in order to get a connection. Contact device provider in order to get this information.</li>
	<li>Use a monitor device specific software to save measurements on the computer. Click &#34;start&#34; to begin recordings and save the results after the end of the measurement. 
	Note: In some devices, data has to be saved live during the measurement. 
	Note: For trouble-shooting take care of the following steps: If the variability of NIRStissue signals is too high, re-evaluate the position of the electrode (avoid bigger venous plexus or arteries directly under the electrodes). High variability of NIRScerebral signals can also be an indirect marker for hyperventilation of divers to reduce partial CO2. Instruct the subject to breath slower and with lower tidal-volumes and re-evaluate the signal. Subjects are allowed to take 3 deep inspirations before final apnea. Avoid including this period into the evaluation of baseline values. The first 30 sec after maximal inspiration are characterized by variable values. Do not use them for analysis.</li>
</ol>
3. Apnea
<ol>
	<li>Have the subjects rest for at least 15 min in a lying position to avoid stress induced changes in blood circulation due to vasoconstriction. Have subjects breathe normally to avoid influences of hyperventilation caused vasoconstriction. Limit the breathing frequency to &#8804; 15 breaths/min.</li>
	<li>Draw blood samples for baseline analysis. Discard the first 5 ml of drawn blood to avoid measurement uncertainty. Flush the catheter after each venous blood collection with sterile saline to prevent clotting.</li>
	<li>Ensure that monitor values are invisible to subjects to avoid visual influences to their apneic performance.</li>
	<li>Check each device for functionality and signal quality. Ensure that electrodes cannot be removed by involuntary movements of the test subject at the end of apnea.</li>
	<li>Conclude with clear agreements. Give a countdown of the last 2 min verbally. Subjects should breathe normally during this preparation time. Prior to the final breath 3 deep inspirations are allowed. Ask the subject to indicate the last inhalation by finger sign. Apnea should be performed as long as possible. 
	Note: The end of the final breath indicates the start of apnea. The end of apnea is defined as the first inspiration after apnea.</li>
	<li>Mark important events (i.e., beginning and end of apnea) electronically to avoid inaccuracies in further time analysis by pressing the &#34;Event Mark Button&#34; on the NIRS device. 
	Note: Movements of the chest and stomach induced by involuntary diaphragm activities are common in the second half of apnea and indicate the struggle phase.</li>
	<li>Draw blood samples at different time-points depending on the aim of the study.</li>
	<li>Centrifuge blood samples at 1,500 x g for 10 min. Take the supernatant and store it at -80 &#176;C for future analysis.</li>
</ol>
4. Processing Data
<ol>
	<li>Processing data from the monitor device:
	<ol>
		<li>Open the saved file on the computer and press &#34;start&#34; to analyze data.</li>
		<li>Click &#34;review&#34; to get access to the trend monitor and select &#34;options&#34; and then &#34;tools&#34; in the MENU submask. Time interval can be changed via &#34;trend interval&#34; if necessary.</li>
		<li>Select the mask &#34;trends&#34; and save. Open file &#34;trends&#34; in a spreadsheet program for further processing.</li>
	</ol>
	</li>
	<li>Processing data from NIRS device:
	<ol>
		<li>Open the software on the computer and connect the NIRS device via WIFI.</li>
		<li>Transfer the data from the NIRS device to the computer.</li>
		<li>Save the data in CSV-format.</li>
		<li>Open file in a spreadsheet program for further processing.</li>
	</ol>
	</li>
</ol>
5. Analyze Values
<ol>
	<li>Create a spreadsheet with both datasets to compare the values. Identify a time interval of at least 30 sec where NIRS-values and SpO2 are constant (&#177; 3%). Take an average of these values to define a baseline-level. 
	Note: Heart rate is known to change considerably prior to apnea. In order to conduct further analysis, a baseline heart rate is defined at a time point 30 sec after initiation of apnea.</li>
	<li>Find the start point of monotonic decrease in rSO2 and SpO2 during apnea by looking for a decrease of values &#62; 2% compared to baseline-levels. This time point is defined as &#34;begin of desaturation&#34;.</li>
	<li>Identify the start point of rSO2 and SpO2 increase at the end of apnea as a monotonic increase of values after termination of apnea. This point is defined as &#34;begin of re-saturation&#34;.</li>
	<li>Calculate the time difference between &#34;start of apnea&#34; and &#34;begin of desaturation&#34; and the time differences between &#34;end of apnea&#34; and &#34;begin of re-saturation&#34; for NIRScerebral, NIRStissue and SpO2. Save each difference in seconds on a separate spreadsheet.</li>
	<li>Optional: Calculate heart rate variability of each participant during the second and the last minute of apnea. This may reveal information about the sympathetic/parasympathetic balance during this stressful phase.</li>
</ol>
6. Statistical Processing
<ol>
	<li>Compare the time differences between &#34;beginning of desaturation&#34; of SpO2, NIRScerebral, and NIRStissue values. Test for Gaussian distribution of the measurement differences (e.g., using Shapiro-Wilk normality test for sample sizes smaller than 50).</li>
	<li>If the distribution of the measurement differences is significantly different from normal distribution, use Wilcoxon signed rank test. If normal distribution can be assumed, consider using paired t-test.</li>
</ol>

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The authors have nothing to disclose.

The total apnea time is mainly caused by lung size and oxygen consumption per minute and influenced by an individuals&#39; ability to withstand the breathing reflex caused by increasing pCO2 or decreasing pO2. Apnea divers are trained to maximize their breath-hold duration and are used to doing so in maximal inspiration. Therefore, the time until hypoxia is detectable differs between individuals and depends on the subject&#39;s physical condition and training status and might even vary by their dail...

Clinically relevant acute hypoxia and concomitant hypercapnia is mostly seen in patients with obstructive sleep apnea syndrome (OSAS), acute airway obstruction or during cardiopulmonary resuscitation. Major limitations in the field of OSAS and other hypoxemic conditions include the limited transferable knowledge about the pathophysiology derived from animal studies and that human models are non-existent 1. To mimic hypoxia in humans, hypoxic gas mixtures have so far been used 2-7. However, these conditions are more representative of high altitude surroundings than of clinical situations where hypoxia, in general, is accompanied by hypercapnia. To monitor tissue oxygenation during cardiac arrest and resuscitation, animal studies have been performed 8 to investigate physiological compensatory mechanisms.
Apneic divers are healthy athletes capable of depressing the breathing impulse that is evoked by low arterial oxygen saturation 9 and an increased pCO2 10,11. We investigated apneic divers in order to mimic clinical situations of acute hypoxia and concomitant hypercapnia 12. This model can be used to evaluate clinical setups, improve the pathophysiological understanding of patients with OSAS or pathological breathing disorders, and reveal new possibilities for studying a potential counter balancing mechanism in cases of apnea. Furthermore, different techniques to detect hypoxia in humans can be tested for feasibility and accuracy in the case of dynamic hypoxia that is present in emergency situations (i.e., airway obstructions, laryngospasm or cannot intubate, cannot ventilate situations) or to simulate intermittent hypoxia in patients with OSAS.
Noninvasive techniques to detect hypoxia in humans are limited. Peripheral pulse oximetry (SpO2) is an approved tool in pre-hospital and hospital settings to detect hypoxia 13. The method is based on light absorption of hemoglobin. However, SpO2 measurement is limited to peripheral arterial oxygenation and cannot be used in cases of pulseless electrical activity (PEA) or centralized minimal circulation 14. In contrast, Near-Infrared Spectroscopy can be used to evaluate cerebral tissue oxygen saturation (rSO2) in real-time during PEA, during hemorrhagic shock or following subarachnoid hemorrhage 15-19. Its use is constantly growing 20 and methodological studies have revealed a positive correlation between SpO2 and rSO2 3,4.
In this study, we provide a model to simulate clinically relevant hypoxia in humans and present a step-by-step methodology to compare peripheral pulse oximetry and NIRS in case of de- and re-saturation. By analyzing physiological data in case of apnea, our understanding of counter balancing mechanisms can be improved.

<table border="0" cellpadding="0" cellspacing="0" width="352">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">SpO2</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">SHP ACC MCABLE-Masimo Set</td>
			<td style="width:88px;">peripheral SpO2-Monitoring</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:88px;">Non Invasive Blood Pressure (NIBP)</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">NIBP cuff M+,&#160; MP00916&#160;</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Electrocardiographic (ECG) &#160;</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">Infinity M540 Monitor</td>
			<td style="width:88px;">ECG monitoring</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Docking station</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">M500 Docking Station</td>
			<td style="width:88px;">connection of M540 to laptop</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:88px;">NIRS</td>
			<td style="width:88px;">NONIN Medical&#8217;s EQUANOX</td>
			<td style="width:88px;">Model 7600 Regional Oximeter System</td>
			<td style="width:88px;">measuring of cerebral and&#160; tissue oxygenation</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:88px;">NIRS diodes</td>
			<td style="width:88px;">EQUANOX Advance Sensor</td>
			<td style="width:88px;">Model 8004CA</td>
			<td style="width:88px;">suited for measuring cerebral and somatic oxygen-saturation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:88px;">Laptop&#160;</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:88px;">DataGrabber</td>
			<td style="width:88px;">Dr&#228;ger Medical AG&#38;CO.KG</td>
			<td style="width:88px;">DataGrabber v2005.10.16</td>
			<td style="width:88px;">software to synchronize M540 with laptop</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:88px;">eVision</td>
			<td style="width:88px;">Nonin Medical. Inc.</td>
			<td style="width:88px;">Version 1.3.0.0</td>
			<td style="width:88px;">software to synchronize NONIN with laptop</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 displays simultaneous recordings of SpO2 and NIRS values (NIRScerebral and NIRStissue) during apnea in one patient. Total apnea time was 363 sec. Following apnea NIRS and SpO2 values remained stable for approximately 140 sec. A decrease in SpO2 was detected after 204 sec by peripheral SpO2 whereas a decrease of NIRScerebral was detected after 238 sec. The lowest measured SpO2 ...

Watch this Scientific Journal Video about A Model to Simulate Clinically Relevant Hypoxia in Humans at JoVE.com

A Model to Simulate Clinically Relevant Hypoxia in Humans

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 ...

a-model-to-simulate-clinically-relevant-hypoxia-in-humans

Research

JoVE Journal

Medicine

8.7K Views.  University Hospital of Bonn. 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).

Video: A Model to Simulate Clinically Relevant Hypoxia in Humans

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

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 Protocol to Acquire the Degenerative Tenocyte from Humans

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical activity and longer life expectancy. Despite its increasing prevalence, the underlying pathogenesis still remains unclear, and treatment is generally symptomatic. Recently, numerous therapeutic options, including growth factors, stem cells, and gene therapy, were investigated in hopes of enhancing the healing potency of the degenerative tendon. However, the majority of these research studies were conducted only on animal models or healthy human tenocytes. Despite some studies using pathological tenocytes, to the best of our knowledge there is currently no protocol describing how to obtain human degenerative tenocytes. The aim of this study is to describe a standard protocol for acquiring human degenerative tenocytes. Initially, the tendon tissue was harvested from a patient with lateral epicondylitis during surgery. Then biopsy samples were taken from the extensor carpi radialis brevis tendon corresponding to structural changes observed at the time of surgery. All of the harvested tendons appeared to be dull, gray, friable, and edematous, which made them visually distinct from the healthy ones. Tenocytes were cultured and used for experiments. Meanwhile, half of the harvested tissues were analyzed histologically, and it was shown that they shared the same key features of tendinopathy (angiofibroblastic dysplasia or hyperplasia). A secondary analysis by immunocytochemistry confirmed that the cultured cells were tenocytes with the majority of the cells having positive stains for mohawk and tenomodulin proteins. The qualities of the degenerative nature of tenocytes were then determined by comparing the cells with the healthy control using a proliferation assay or qRT-PCR. The degenerative tenocyte displayed a higher proliferation rate and similar gene expression patterns of tendinopathy that matched previous reports. Overall, this new protocol might provide a useful tool for future studies of tendinopathy.

In vitro use of degenerative tenocytes is essential when investigating the efficacy of novel treatment on tendinopathy. However, most research studies use only the animal model or a healthy tenocyte. We propose the following protocol to isolate human degenerative tenocytes during surgery.

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic ...