R01HL138932, RO1HL133100, RO1HL128970, AHACDA34700025

All experimental protocols have been approved by the Institutional Animal Care and Use Committee (MO18M211).
1. Leptin Infusion
NOTE: In order to examine the effect of leptin on breathing, we infused leptin subcutaneously in lean C57BL/6J mice by an osmotic pump to raise circulating leptin levels to those observed in obese mice.
<ol>
	<li>Osmotic pump preparation
	<ol>
		<li>Weigh the empty pump to check the net weight of the solution loaded.</li>
		<li>Add Leptin (5 mg/mL) to the osmotic pump (1 &#956;L/h for 3 days). Fill the pump with a small syringe (1 mL). After filling, close the pump with the provided blunt tipped 27 gauge filling tube. 
		NOTE: The syringe and the attached tube should be free of air bubbles.</li>
		<li>After filling, weigh the pump again to check the net weight of the solution.</li>
		<li>Insert the leptin pump subcutaneously in the interscapular area. 
		NOTE: If you want to start the infusion immediately, incubate the pre-filled pump in the sterile saline at 37 &#176;C for at least 4 to 6 h (preferably overnight).</li>
	</ol>
	</li>
</ol>
2. Hypoxic Ventilatory Response (HVR)
NOTE: Thermoneutral conditions eliminate stressful factors imposed by cool ambient temperature and significantly modify metabolism in mice. Therefore, all respiratory measurements should be performed at the thermoneutral conditions (t = 30 &#176;C) using a neonatal incubator12 (Figure 1A). The whole body plethysmography chamber (WBP) has been used for all measurements. All animals should be acclimated to the barometric plethysmography chamber and to a sham neck collar for subsequent pulse oximetry recording for 3-5 days prior to the HVR measurements. HVR is recorded between 10 AM and 5 PM. HVR is suppressed during sleep, therefore it can be measured separately during sleep and quiet wakefulness13. To ensure the specific sleep-wake stage of the animal during the HVR measurement, EEG/EMG electrodes should be implanted as an EEG/EMG headmount as previously described14. Animals should be allowed to recover for at least 72 h after the headmount. Sleep staging should be performed in 5 s epochs. NREM sleep is recognized by the slow wave EEG activity occupying greater than 50% of the epoch. Quiet wakefulness is manifested by the alpha EEG activity in the absence of movements. REM sleep is identified by the predominant alpha and theta EEG activity in the presence of reduced muscle tone on EMG. Typically, REM sleep is not observed during the HVR gas challenges.
<ol>
	<li>The HVR recording protocol
	<ol>
		<li>Use the WBP chamber of the following size: internal diameter of 80 mm, height of 50.5 mm, and volume of 0.4 L. The WBP chamber consists of two chambers, sealed and reference chambers, and a circular platform to place the mouse14 (Figure 1B). Inflow and outflow inside the chamber are controlled by positive and negative pressure sources maintaining the atmospheric pressure. This control creates a steady bias flow in the chamber and prevents CO2 retention. For more details about the WBP set up, see Hernandez and colleagues15.</li>
		<li>Measure the temperature inside the chamber and the relative humidity in the room prior to placing the mouse in the WBP.</li>
		<li>Measure the body weight and rectal temperature.</li>
		<li>Put the oximeter collar around the neck of the mouse (the area should be previously shaved).</li>
		<li>Place the mouse inside the WBP and ensure that the chamber is completely sealed in order to avoid air leakages.</li>
		<li>Wait for approximately 30 min until the mouse is quiet and the chamber is at a constant volume.</li>
		<li>Normoxia: after 30 min, if the mouse is quiet, start recording the respiratory signals and peripheral oxygen saturation (SpO2) at normoxia phase (21% FiO2 at 1 atm of pressure) for 20 min, using LabChart 7 Pro (version 7.2).</li>
		<li>Hypoxia: after 20 min of quiet normoxia, start recording the first cycle of acute hypoxia and SpO2. For hypoxia phase, expose the animals to a constant mixed gas flow composed by 10% O2 and 3% CO2 for 5 min.
		<ol>
			<li>During the first 30 sec of hypoxia, the mixed gas flows through the chamber via 2 small side ports at the base allowing the FiO2 to drop from 21% (at 1 atm of pressure) to 10%.</li>
			<li>After the initial 30 s, close one of the small side ports of the WBP chamber and keep recording at the constant hypoxia at 10% FiO2 for 5 min. 
			NOTE: The mixture of 10% O2 and 3% CO2 at hypoxia is used in order to maintain eucapnea11.</li>
		</ol>
		</li>
		<li>After the 5 min of hypoxia, expose the mouse to room air again (21% FiO2 at 1 atm of pressure) by switching the inflow source.</li>
		<li>Wait for at least 30 min until the next normoxia recording in order to recover from the previous hypoxic exposure to avoid the ventilatory roll-off phenomenon (i.e., central suppression of breathing during hypoxia16). 
		NOTE: Repeat normoxia/hypoxia cycles three times in each mouse to assure reproducibility of the measurements. In our experience, additional (greater than 3 times on a given day) hypoxic exposures should be avoided, because of the ventilatory adaptation (the roll-off phenomenon).</li>
		<li>At the end of the experiment, calibrate the WBP chamber (with the mouse is still inside) by injecting 1 mL of room air 3 times with a syringe plugged in one of the small side ports at the base of the chamber.</li>
		<li>Measure the temperature in the chamber again with the animal inside it.</li>
		<li>Open the chamber and measure the rectal temperature of the mouse before placing it back to its home cage.</li>
	</ol>
	</li>
	<li>HVR calculation
	<ol>
		<li>Digitize all signals for HVR calculation at 1,000 Hz (sampling frequency per channel). 
		NOTE: WBP tidal volume analysis is performed in Lab Chart 7 based on Drorbaugh and Fenn equation17. Required variables comprise mouse rectal temperature and the chamber temperature (before and after HVR measurement), relative humidity, and the chamber gas constant. This constant is resultant from WBP pressure deflection after the 1 mL injections of air in the calibration phase. For more details, see Hernandez and colleagues15.</li>
		<li>After calculating the Drorbaugh and Fenn equation, multiply the tidal volume (Vt) chamber channel by the constant.</li>
		<li>Selection of recordings for the analysis
		<ol>
			<li>Normoxia: select only sections of steady breathing with constant tidal volume. Avoid sections in close proximity to mouse&#8217;s movements.</li>
			<li>Hypoxia: discard first 30 s when O2 levels are declining from 21% to 10%, select sections from 30 s to 2 min of 10% O2 exposure (a 90 s interval). Within this interval, select only sections with steady breathing with constant tidal volume. Avoid sections in close proximity to mouse&#8217;s movements. The analysis is reliable if at least 10 s of hypoxia is selected in each cycle. 
			NOTE: The peripheral component of chemoreflex governed by CB predominates during the first 1-2 min of exposure16,18,19. During the second phase, between 2 min and 5 min of hypoxic exposure, both peripheral and central component play a role. Finally, the third phase, &#62; 5 min, is characterized by hypoventilation (the roll-off phenomenon) governed predominantly by central chemoreceptors. Our experience shows that mice are often awake during hypoxic exposition because of the manual switches in the airflow source.</li>
		</ol>
		</li>
		<li>After the selection, calculate the mean minute ventilation (VE) at normoxic and hypoxic conditions using the formula VE = respiratory rate X tidal volume.</li>
		<li>Calculate the mean SpO2 at normoxia and hypoxia conditions.</li>
		<li>Calculate the HVR manually using the formula HVR = (VE (10% O2) - VE (21%O2)) / (SpO2 (10% O2) &#8211; SpO2 (21% O2)). 
		NOTE: In C57BL/6J mice, the osmotic pump for leptin infusion may impair the accurate SpO2 signal detection by the neck collar. In this case, HVR is calculated based on the FiO2 values at normoxic and hypoxic conditions as HVR = &#916;VE / &#916;FiO211.</li>
	</ol>
	</li>
</ol>
3. Carotid Body Denervation or Carotid Sinus Nerve Dissection (CSND)
NOTE: We performed combined surgical and chemical denervation one week apart, because surgical denervation alone does not abolish the hypoxic chemoreflex.
<ol>
	<li>Surgical preparation
	<ol>
		<li>Perform all the procedures on adult male C57BL/6J mice. Sterilize all surgical instruments. Use sterile surgical gloves, syringes and cotton tipped applicators.</li>
		<li>Anesthetize male C57BL/6J mice with 1-2% isoflurane using a nose cone and place a warm blanket to prevent hypothermia. Isoflurane is carefully titrated to maintain respiratory rate at 1 Hz (60 breaths/min). The adequacy of anesthesia prior to starting surgeries will be assessed by the breathing frequency, the absence of movements and audible noises, the absence of response to tactile stimuli and the forelimb or hindlimb pedal withdrawal reflex.</li>
		<li>Administer Buprenorphine (0.05 mg/kg) intraperitoneally to prevent pain discomfort. Remove the hair at ventral region of the neck and disinfect the area with betadine and alcohol.</li>
		<li>To prevent corneal desiccation, lubricate the mice&#8217;s eyes with sterile ophthalmic ointment during anesthesia.</li>
	</ol>
	</li>
	<li>CSND procedures 
	Stage 1: Surgical denervation
	<ol>
		<li>After midline incision, expose the bifurcation of the common carotid arteries by removing connective tissues and adipose tissue.</li>
		<li>Identify the hypoglossal nerve, which is usually very prominent, and then lift it up to expose the glossopharyngeal nerve immediately underneath. Dissect carotid sinus nerves bilaterally using micro spring scissors.</li>
		<li>Close the incision with 6-0 silk suture.</li>
		<li>Administer 1 mL of normal saline subcutaneously to prevent dehydration.</li>
		<li>House the mice in a recovery chamber and monitor their behavior every 15 min for initial 1 h until the mice regain consciousness to maintain sternal recumbency. Return the mice to their home cages after they are fully recovered. Keep monitoring the mice twice per day for next 3 days. Give additional buprenorphine if mice show any signs of pain (e.g., reduced appetite, restlessness). 
		Stage 2: Chemical denervation</li>
		<li>One week after the surgery, paint the carotid artery with a solution of 2% phenol diluted in ethanol at the points of branching from the glossopharyngeal nerve to the cranial pole of the CB. The same post-operative care should be provided after the chemical denervation as described above in the surgical denervation section. 
		NOTE: For a sham surgery group, the same procedures are performed except the carotid sinus nerve dissection.</li>
		<li>Let the animals recover for 5-7 days before HVR measurements.</li>
	</ol>
	</li>
</ol>
4. Expression of LepRb in the CB using an adenoviral vector (Ad-LepRb) vs control (Ad-LacZ)
<ol>
	<li>Ad-LepRb and Ad-LacZ suspension
	<ol>
		<li>Transfect the mice with adenovirus (Ad-LepRb-GFP, 2-5x1010 pfu/mL for overexpression, Ad-Lacz, 1 x 1010 pfu/mL for control) to the CB area.</li>
		<li>Thaw the Matrigel matrix by submerging the vial in ice in a 4 &#176;C refrigerator overnight.</li>
		<li>Gently suspend adenovirus in liquid Matrigel matrix at 1:5 (1 &#956;L of Matrigel matrix and 4 &#956;L of adenovirus applied bilaterally).</li>
		<li>Always keep viral suspension on ice until it is applied in the CB.</li>
	</ol>
	</li>
	<li>Surgical preparation
	<ol>
		<li>Use the same surgical instruments as the CSND protocol.</li>
		<li>Anesthetize LepRb-deficient db/db mice with 2-2.5% isoflurane using a nose cone and place the animals on a warm blanket to prevent hypothermia. Isoflurane is carefully titrated to maintain respiratory rate at 1 Hz (60 breaths/min). The adequacy of anesthesia will be assessed by the breathing frequency, the absence of movements and audible noises, the absence of response to tactile stimuli and the forelimb or hindlimb pedal withdrawal reflex.</li>
		<li>Administer buprenorphine (0.05 mg/kg) intraperitoneally.</li>
		<li>Remove the hair at ventral region of the neck and disinfect the area with betadine and alcohol as in the CSND protocol.</li>
		<li>To prevent corneal desiccation, lubricate the mice&#8217;s eyes with sterile ophthalmic ointment during anesthesia.</li>
	</ol>
	</li>
	<li>Adenovirus treatment of the CB
	<ol>
		<li>Expose CBs as described in the CSND protocol and apply 5 &#956;L of the viral suspension to the CB area bilaterally.</li>
		<li>Wait 2-3 min until the liquid Matrigel matrix becomes a gel. After confirming that the viral suspension was congealed, close the incision with 6.0 silk suture.</li>
	</ol>
	</li>
	<li>After surgery
	<ol>
		<li>House the mice in a recovery chamber and monitor their behavior every 15 min for initial 1 h until the mice regain consciousness to maintain sternal recumbency. Return the mice to their home cages after they are fully recovered. Keep monitoring the mice twice per day for next 3 days. Give additional buprenorphine if mice show any signs of pain (e.g., reduced appetite, restlessness).</li>
		<li>Administer Buprenorphine (0.05 mg/kg) intraperitoneally as needed to prevent pain discomfort during the post-operative period.</li>
		<li>Measure HVR 9 days after the adenovirus transfection.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicting interests or disclosures.

The main focus of our study was to examine respiratory effects of leptin signaling in the CB. Several protocols have been developed to assess the role of leptin in a mechanistic fashion. First, specific contribution of CB to the HVR was analyzed by careful quantification of the HVR during the first 2 min of hypoxic exposure. Second, the relevance of CB in leptin-mediated up-regulation of control of breathing was examined by two complementary approaches. In lean wild-type mice with low leptin levels, the HVR was measured ...

An adipocyte produced hormone leptin acts in the hypothalamus to suppress food intake and increase metabolic rate. Studies performed in our laboratory1,2 and by other investigators3,4 showed that leptin increases the hypercapnic ventilatory response (HVR) preventing obesity hypoventilation in leptin deficient obesity. However, a majority of obese individuals have high plasma leptin levels and demonstrate resistance to the metabolic and respiratory effects of the hormone5,6,7,8. Resistance to leptin is multifactorial, but limited permeability of the blood-brain barrier (BBB) to leptin plays a major role. We propose that leptin acts below BBB in a key organ of peripheral hypoxic sensitivity, the carotid bodies (CB), to defend breathing in obese individuals. CBs express the long functional isoform of leptin receptor, LepRb, but the role of CB in respiratory effects of leptin has not been sufficiently elucidated9,10.
The goal of our method was to examine the effect of leptin signaling in the CB on HVR. Our rationale was to perform (a) loss of function experiments infusing leptin in mice with intact carotid bodies and denervated carotid bodies followed by HVR measurements; (b) gain of function experiments in db/db mice lacking LepRb, in which we measured the HVR at baseline and after expression of LepRb exclusively in CB. The advantage of our techniques was that we performed all our experiments in unrestrained unanesthetized mice during sleep and wakefulness. Previous investigators either performed their experiments under anesthesia9 or did not measure effects of leptin during sleep10. In addition, our study is the first to utilize a unique gain of function approach with selective LepRb expression in CB described above.
In the broad context, our approach can be generalized to other receptors expressed in CB and their role in hypoxic sensitivity. Investigators can infuse a ligand to a receptor of interest and measure the HVR at baseline and after CB denervation. As a complementary approach, a receptor of interest can be overexpressed in CB and HVR measurements can be performed before and after overexpression using our technology described in this manuscript.

<table>
	<tbody>
		<tr>
			<td>1ml Insulin Syringes</td>
			<td>BD Biosciences</td>
			<td>309311</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS (pH 7.4)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Ad-Lacz</td>
			<td>Dr. Christopher Rhodes (University of Chicago)</td>
			<td></td>
			<td>1x1010 pfu/ml</td>
		</tr>
		<tr>
			<td>Ad-LepRb-GFP</td>
			<td>Vector Biolabs</td>
			<td>ADV-263380</td>
			<td>2-5x1010 pfu/ml</td>
		</tr>
		<tr>
			<td>Anesthetic cart</td>
			<td>Atlantic Biomedical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Products Ltd.</td>
			<td>12496-0757-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Buprenex)</td>
			<td>Reckitt Benckiser Healthcare Ltd.</td>
			<td>12496-0757-5</td>
			<td>0.3mg/ml</td>
		</tr>
		<tr>
			<td>C57Bl/6J</td>
			<td>Jackson laboratory</td>
			<td>000664</td>
			<td>Mice Strain</td>
		</tr>
		<tr>
			<td>Cotton Gauze Sponges</td>
			<td>Fisherbrand</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr>
			<td>db/db</td>
			<td>Jackson laboratory</td>
			<td>000697</td>
			<td>Mice Strain</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Vetone</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Chart</td>
			<td>Data Science International (DSI)</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Spring Scissors</td>
			<td>World Precision Instruments (WPI)</td>
			<td>14124</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Ox Plus</td>
			<td>STARR Life Sciences Corp.</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Mouse Ox Plus Collar Sensor</td>
			<td>STARR Life Sciences Corp.</td>
			<td>015022-2</td>
			<td>Medium Collar Clip Special 7&#8221;</td>
		</tr>
		<tr>
			<td>Mouse Whole Body Plethysmography Chamber</td>
			<td>Data Science International (DSI)</td>
			<td>PLY3211</td>
			<td></td>
		</tr>
		<tr>
			<td>Ohio Care Plus Incubator</td>
			<td>Ohmeda</td>
			<td>HCHD000173</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors</td>
			<td>World Precision Instruments (WPI)</td>
			<td>501753-G</td>
			<td>Straight</td>
		</tr>
		<tr>
			<td>Osmotic Pump</td>
			<td>Alzet</td>
			<td>1003D</td>
			<td>1ul per hour, 3 days</td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma-Aldrich</td>
			<td>P4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Leptin protein</td>
			<td>R&#38;D systems</td>
			<td>498-OB-05M</td>
			<td>5mg</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>RICCA Chemical</td>
			<td>7210-16</td>
			<td>0.9% Sodium Chloride</td>
		</tr>
		<tr>
			<td>Sterile Surgical Suture</td>
			<td>DemeTech</td>
			<td>DT-639-1</td>
			<td>Silk, size 6-0</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Innovative Calibration Solutions (INNOCAL)</td>
			<td>EW 20250-91</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental approach to examine leptin signaling in the carotid bodies and its effects on control of breathing

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The carotid bodies (CB), a key organ of peripheral hypoxic sensitivity, express the long functional isoform of leptin receptor (LepRb) but the role of leptin signaling in control of breathing has not been fully elucidated. We examined the hypoxic ventilatory response (HVR) (1) in C57BL/6J mice before and after leptin infusion at baseline and after CB denervation; (2) in LepRb-deficient obese db/db mice at baseline and after LepRb overexpression in CBs. In C57BL/6J mice, leptin increased HVR and effects of leptin on HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB augmented the HVR. Therefore, we conclude that leptin acts in CB to augment responses to hypoxia.

Continuous infusion of leptin significantly increased HVR in lean C57BL/6J mice from 0.23 to 0.31 mL/min/g/&#916;FiO2 (P &#60; 0.001, Figure 2)11. CSND abolished the leptin-induced increase in HVR (Figure 2), while no attenuating effects of CSND on HVR were observed in sham surgery group after leptin infusion.
LepRb expression in the CB of LepRb-deficient obese...

Watch this Scientific Journal Video about Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing at JoVE.com

Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing

Our study focuses on the effects of leptin signaling in carotid body (CB) on the hypoxic ventilatory response (HVR). We performed &#39;loss of function&#39; experiments measuring the effect of leptin on HVR after CB denervation and &#39;gain of function&#39; experiments measuring HVR after overexpression of the leptin receptor in CB.

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The ...

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5.8K Views.  Johns Hopkins University. Our study focuses on the effects of leptin signaling in carotid body (CB) on the hypoxic ventilatory response (HVR). We performed 'loss of function' experiments measuring the effect of leptin on HVR after CB denervation and 'gain of function' experiments measuring HVR after overexpression of the leptin receptor in CB.

Video: Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...