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.
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.
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.
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.
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 °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.
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.
4. Expression of LepRb in the CB using an adenoviral vector (Ad-LepRb) vs control (Ad-LacZ)
Continuous infusion of leptin significantly increased HVR in lean C57BL/6J mice from 0.23 to 0.31 mL/min/g/ΔFiO2 (P < 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 db/db mice induced a significant increase in HVR from 0.05 to 0.06 mL/min/g/ΔSpO2 (Figure 3). In animals transfected with control Ad-LacZ in CB, HVR did not change.
Figure 1: HVR measurements. Experiments should be performed at thermoneutral conditions using (A) a neonatal incubator at 30 °C and are recorded in a (B) whole body plethysmography chamber. Please click here to view a larger version of this figure.
Figure 2: Leptin augmented the hypoxic ventilatory response (HVR) and the effects were abolished by carotid sinus nerve dissection (CSND) in C57BL/6J mice. This figure has been modified from Caballero-Eraso et al.11. Please click here to view a larger version of this figure.
Figure 3: LepRb expression in the carotid bodies (CB) of LepRb-deficient db/db mice increased the hypoxic ventilatory response (HVR). This figure has been modified from Caballero-Eraso et al.11. Please click here to view a larger version of this figure.
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 at baseline and after continuous infusion of leptin; the experiment was repeated after CB denervation. In LepRb-deficient db/db mice, the HVR was measured at baseline and after LepRb expression in the CB.
Several protocols have been used to measure HVR in rodents, exposing the animals to hypoxic gases. We have developed an HVR protocol to examine the role of CB in the peripheral chemoreflex and the effects of leptin in CB on the control of breathing. CB chemoreflex has a relatively short time domain. The first 1-2 min of hypoxia are characterized by an acute augmentation of ventilatory response followed by a slow return to baseline ventilation after 2 min of the carotid sinus nerve stimulation16,18,19. Thus, in our HVR protocol, the analyses are conducted within a 90 s interval between the first 30 s of hypoxia and 2 min of 10% O2 exposure, corresponding to the domination of peripheral chemoreflex governed by CB. This short-time analysis avoids the hypoxic ventilatory depression in the animals. In our HVR protocol, we also used a fixed 3% CO2 tension in hypoxia in order to circumvent the hypocapnia induced by hyperventilation during acute hypoxic exposure11. Finally, we have developed the HVR protocol under constant thermoneutral conditions, keeping the mice at a temperature of around 30 °C. Our experience shows that short exposures to hypoxia can decrease rectal temperature in mice if exposure occurs at room temperature11, while hypoxia at thermoneutrality does not induce significant metabolic changes12.
CSND in mice is technically challenging, because of the small size of the animals and their CBs. We have developed a consistently successful approach with nearly 100% survival rate by strict adherence to our protocol. Controlled conditions in our protocol includes thermoneutral environment, carefully controlled anesthesia and standard sterile microsurgical techniques with visualization of the glossopharyngeal nerve as vigilant post-operative management with pain control. Our experience shows that surgical denervation alone does not abolish the hypoxic chemoreflex. The second step, chemical denervation, is also followed by careful post-operative management to improve survival.
Our most innovative technique is selective gene over-expression in the CB area. This approach has not been implemented previously, because of the small size of the CBs and the lack of specific drivers allowing to express a gene of interest in a particular cell type. In fact, type I CB cells are very similar to sympathetic neurons or cells of the adrenal medulla, whereas type II cells are similar to astrocytes20,21. We took advantage of db/db mice, which lack the LepRb gene, our ability to apply adenoviral suspension almost exclusively to the CB area, and properties of the Matrigel matrix, which rapidly solidifies at 37 °C. Our novel approach can be used in the future to study a role of any gene expressed in CB using mice with the whole body tyrosine hydroxylase specific (type I cells) or GFAP specific (type II cells) knockouts.
Our protocols have several limitations. First, we used 3% CO2 to determine the HVR and the question remains open if the fraction of the HVR can actually be attributed to the hypercapnic response. In order to address this limitation, investigators can simultaneously measure responses to 3% CO2 balanced in hyperoxic gas, which would turn the CB off. Second, HVR may not be completely eliminated by CSND22. This phenomenon can be attributed to neuroplasticity, which is particularly prominent in mice. Therefore, it is important to study HVR as soon as possible after CSND and always use sham surgery control. Third, our CB gene expression approach lacked cell type and organ specificity. Molecular techniques with the future use of more selective promoters may help to counter this limitation.
In conclusion, despite described above limitations, our protocols in combination allow to study the role of specific CB genes in physiological responses to hypoxia.
The authors have nothing to disclose.
R01HL138932, RO1HL133100, RO1HL128970, AHACDA34700025
1ml Insulin Syringes | BD Biosciences | 309311 | |
1x PBS (pH 7.4) | Gibco | 10010-023 | 500 ml |
Ad-Lacz | Dr. Christopher Rhodes (University of Chicago) | 1×1010 pfu/ml | |
Ad-LepRb-GFP | Vector Biolabs | ADV-263380 | 2-5×1010 pfu/ml |
Anesthetic cart | Atlantic Biomedical | ||
Betadine | Purdue Products Ltd. | 12496-0757-5 | |
Buprenorphine (Buprenex) | Reckitt Benckiser Healthcare Ltd. | 12496-0757-5 | 0.3mg/ml |
C57Bl/6J | Jackson laboratory | 000664 | Mice Strain |
Cotton Gauze Sponges | Fisherbrand | 22-362-178 | |
db/db | Jackson laboratory | 000697 | Mice Strain |
Ethanol | Pharmco-AAPER | 111000200 | |
Isoflurane | Vetone | 502017 | |
Lab Chart | Data Science International (DSI) | Software | |
Matrigel Matrix | BD Biosciences | 356234 | |
Micro Spring Scissors | World Precision Instruments (WPI) | 14124 | |
Mouse Ox Plus | STARR Life Sciences Corp. | Software | |
Mouse Ox Plus Collar Sensor | STARR Life Sciences Corp. | 015022-2 | Medium Collar Clip Special 7” |
Mouse Whole Body Plethysmography Chamber | Data Science International (DSI) | PLY3211 | |
Ohio Care Plus Incubator | Ohmeda | HCHD000173 | |
Operating Scissors | World Precision Instruments (WPI) | 501753-G | Straight |
Osmotic Pump | Alzet | 1003D | 1ul per hour, 3 days |
Phenol | Sigma-Aldrich | P4557 | |
Recombinant Mouse Leptin protein | R&D systems | 498-OB-05M | 5mg |
Saline | RICCA Chemical | 7210-16 | 0.9% Sodium Chloride |
Sterile Surgical Suture | DemeTech | DT-639-1 | Silk, size 6-0 |
Thermometer | Innovative Calibration Solutions (INNOCAL) | EW 20250-91 |