Unanesthetized Adult Rats Research Articles

Evidence for the emergence of an opioid‐resistant respiratory rhythm following fentanyl overdose

Breathing resumes within one to two minutes following fentanyl overdose induced apnea in spontaneously breathing rats. As this regular rhythm is produced at a time wherein fentanyl concentrations and receptor occupancy are likely to be extremely high, the mechanisms initiating and sustaining such a respiratory activity remain unclear.Forty-four un-anesthetized adult rats were studied in an open-flow plethysmograph. Regardless of the dose of fentanyl that was used, i.e. 50 μg.kg−1 (n = 8), 100 μg.kg−1 (n = 8) or 300 μg.kg−1 (n = 7), all rats developed an immediate central apnea followed by a depressed regular rhythm that was produced 118, 97 and 81 s (median) later, respectively. Only one rat did not recover. This inspiratory and regular activity consisted of a low frequency and tidal volume pattern with a significant reduction in V̇E/V̇CO2 ratio, which persisted for at least 30 min and that was not different between 100 or 300 μg.kg−1. The time at which this respiratory rhythm emerged, following the highest dose of fentanyl, was not affected by 100 % O2 or 8% CO2/15 % O2. The absolute level of ventilation was however higher in hypercapnic and moderately hypoxic conditions than in hyperoxia. When a second injection of the highest dose of fentanyl (300 μg.kg−1) was performed at 10 min, ventilation was not significantly affected and no apnea was produced in major contrast to the first injection. When a similar injection was performed 30 min after the first injection, in a separate group of rats, an apnea and breathing depression was produced in 30 % of the animals, while in the other rats, ventilation was unaffected. We conclude that the depressed regular respiratory activity emerging during and following fentanyl overdose is uniquely resistant to fentanyl.

Patterns of cardiovascular variability after long-term sino-aortic denervation in unanesthetized adult rats

Baroreflex dysfunction is a diffuse chronic condition that is expected to be followed by a profound loss of organization of BP and HR variability. Nevertheless, long-term effects of baroreflex withdrawal are still debated. Aim of our work was to study BP and HR changes long term after sino-aortic denervation (SAD). Inter-beat-interval (IBI) and intra-arterial BP were recorded beat-by-beat in 43 Wistar-Kyoto rats (Controls, n = 33; SAD rats, n = 10). Power spectra were calculated in controls and in SAD rats within three days and at seven months from denervation. Compared to controls, chronic SAD rats showed 1) similar mean BP (control vs SAD: 95 ± 16 vs 87 ± 22 mmHg) and IBI (171 ± 22 vs 181 ± 15 ms) values, 2) dramatically higher values of BP variance (12 ± 2 vs 64 ± 2 mmHg2, p < 0.01) and of ultra- (ULF) and very-low-frequency (VLF) BP oscillations, 3) dramatically higher values of IBI variability (24 ± 2 vs 71 ± 4 ms2, p < 0.01) and of ULF-IBI oscillations that were synchronized with BP oscillations. Chronic SAD rats reveal a marked change in the pattern of cardiovascular variability characterized by the appearance of synchronized slower oscillations of BP and HR. The cardiovascular system, therefore, retains a high level of organization despite the absence of a reflex control mechanism.

Neurochemical and electrical modulation of the locus coeruleus: contribution to CO2drive to breathe.

The locus coeruleus (LC) is a dorsal pontine region, situated bilaterally on the floor of the fourth ventricle. It is considered to be the major source of noradrenergic innervation in the brain. These neurons are highly sensitive to CO2/pH, and chemical lesions of LC neurons largely attenuate the hypercapnic ventilatory response in unanesthetized adult rats. Developmental dysfunctions in these neurons are linked to pathological conditions such as Rett and sudden infant death syndromes, which can impair the control of the cardio-respiratory system. LC is densely innervated by fibers that contain glutamate, serotonin, and adenosine triphosphate, and these neurotransmitters strongly affect LC activity, including central chemoreflexes. Aside from neurochemical modulation, LC neurons are also strongly electrically coupled, specifically through gap junctions, which play a role in the CO2 ventilatory response. This article reviews the available data on the role of chemical and electrical neuromodulation of the LC in the control of ventilation.

The Ca2+‐activated K+ channels play a braking role in locus coeruleus neurons during CO2 exposure (872.1)

The LC has an important role in central chemoreception and K+ channels have been suggested to be sensitive to changes of pH. Studies have shown that some ion channels present in the cell membrane of LC neurons, among them the Ca2+‐activated K+ channels (KCa), are activated by high levels of CO2. These channels appear to hyperpolarize LC neurons and limit increased firing rate in response to hypercapnic acidosis. We evaluated the participation of KCa in the CO2‐drive to breath in unanesthetized adult rats by bilaterally microinjecting Paxiline (KCa blocker, 100µM or 500µM / 0.1µL) or vehicle (aCSF) intra‐LC. Bilateral KCa blockade in LC neurons did not affect resting ventilation. However, the increase in ventilation produced by hypercapnia (7% CO2) was amplified by 39 and 33% after Paxilline (100µM or 500µM, respectively) injection (1305.2 ± 164.2 mL.kg‐1.min‐1 in vehicle group vs 1822.0 ± 201.7 in Paxilline 100µM group and 1735.3 ± 116.5 in Paxilline 500µM group) due largely to a increase in tidal volume. The results suggest that KCa++ modulate the hypercapnic ventilatory response as braking pathway, but that they are not involved in respiratory control during normocapnia, in the LC of adult rats.Grant Funding Source: FAPESP and CNPq

Cardiorespiratory effects of gap junction blockade in the locus coeruleus in unanesthetized adult rats

The locus coeruleus (LC) plays an important role in central chemoreception. In young rats (P9 or younger), 85% of LC neurons increase firing rate in response to hypercapnia vs. only about 45% of neurons from rats P10 or older. Carbenoxolone (CARB – gap junction blocker) does not affect the % of LC neurons responding in young rats but it decreases the % responding by half in older animals. We evaluated the participation of gap junctions in the CO2 ventilatory response in unanesthetized adult rats by bilaterally microinjecting CARB (300μM, 1mM or 3mM/100nL), glycyrrhizic acid (GZA, CARB analog, 3mM) or vehicle (aCSF – artificial cerebrospinal fluid) into the LC of Wistar rats. Bilateral gap junction blockade in LC neurons did not affect resting ventilation; however, the increase in ventilation produced by hypercapnia (7% CO2) was reduced by ∼25% after CARB 1mM or 3mM injection (1939.7±104.8mLkg−1min−1 for the aCSF group and 1468.3±122.2mLkg−1min−1 for 1mM CARB, P<0.05; 1939.7±104.8mLkg−1min−1 for the aCSF group and 1540.9±68.4mLkg−1min−1 for the 3mM CARB group, P<0.05) due largely to a decrease in respiratory frequency. GZA injection or CARB injection outside the LC (peri-LC) had no effect on ventilation under any conditions. The results suggest that gap junctions in the LC modulate the hypercapnic ventilatory response of adult rats.

Quantifying conversion of linoleic to arachidonic and other n-6 polyunsaturated fatty acids in unanesthetized rats

Isotope feeding studies report a wide range of conversion fractions of dietary shorter-chain polyunsaturated fatty acids (PUFAs) to long-chain PUFAs, which limits assessing nutritional requirements and organ effects of arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3) acids. In this study, whole-body (largely liver) steady-state conversion coefficients and rates of circulating unesterified linoleic acid (LA, 18:2n-6) to esterified AA and other elongated n-6 PUFAs were quantified directly using operational equations, in unanesthetized adult rats on a high-DHA but AA-free diet, using 2 h of intravenous [U-(13)C]LA infusion. Unesterified LA was converted to esterified LA in plasma at a greater rate than to esterified gamma-linolenic (gamma-LNA, 18:3n-6), eicosatrienoic acid (ETA, 20:3n-6), or AA. The steady-state whole-body synthesis-secretion (conversion) coefficient k*(i) to AA equaled 5.4 x 10(-3) min(-1), while the conversion rate (coefficient x concentration) equaled 16.1 micromol/day. This rate exceeds the reported brain AA consumption rate by 27-fold. As brain and heart cannot synthesize significant AA from circulating LA, liver synthesis is necessary to maintain their homeostatic AA concentrations in the absence of dietary AA. The heavy-isotope intravenous infusion method could be used to quantify steady-state liver synthesis-secretion of AA from LA under different conditions in rodents and in humans.

Systemic inflammation suppresses hypoxic and hypercapnic ventilatory responses in rats.

Chemoreflex responses to hypoxia and hypercapnia are critical elements of ventilatory control. However, chemoreflexes may be undermined by factors frequently associated with lung disease, such as systemic inflammation. Here, we tested the hypothesis that systemic inflammation suppresses ventilatory responses to hypoxia and hypercapnia in rats. Ventilatory responses were assessed in unanesthetized adult rats via whole‐body plethysmography 1 day after lipopolysaccharide administration (LPS: 1 mg in saline, i.p.). In Lewis rats, LPS suppressed both hypoxic (10.5% O2; vehicle: 90±19%; LPS: 37±11%; p &lt; 0.05) and hypercapnic ventilatory responses (7% CO2; vehicle: 223±22%; LPS: 66±26%; p &lt; 0.05). Splenic iNOS mRNA increased 6.5‐fold (qPCR) in LPS‐treated Lewis rats, demonstrating systemic inflammation. In contrast, there was no evidence for suppressed hypoxic (vehicle: 127±14%; LPS: 92±21%) or hypercapnic ventilatory responses (vehicle: 216±19%; LPS: 239±27%) in LPS‐treated Sprague Dawley rats, nor were splenic iNOS mRNA levels increased. Thus, although LPS sensitivity varies among rat strains, systemic inflammation suppressed chemoreflex responses to hypoxia and hypercapnia. Suppressed chemoreflex responses may impair the ability to respond appropriately to ventilatory challenges in disease states associated with inflammation, including many lung diseases (NIH HL69064 and HL007654).Grant Funding Source: NIH HL69064 and HL007654

Ventilation and phrenic output following high cervical spinal hemisection in male vs. female rats

Female sex hormones influence the neural control of breathing and may impact neurologic recovery from spinal cord injury. We hypothesized that respiratory recovery after C2 spinal hemisection (C2HS) differs between males and females and is blunted by prior ovariectomy (OVX) in females. Inspiratory tidal volume (VT), frequency (fR), and ventilation (VE) were quantified during quiet breathing (baseline) and 7% CO2 challenge before and after C2HS in unanesthetized adult rats via plethysmography. Baseline breathing was similarly altered in all rats (reduced VT, elevated fR) but during hypercapnia females had relatively higher VT (i.e. compared to pre-injury) than male or OVX rats (p<0.05). Phrenic neurograms recorded in anesthetized rats indicated that normalized burst amplitude recorded ipsilateral to C2HS (i.e. the crossed phrenic phenomenon) is greater in females during respiratory challenge (p<0.05 vs. male and OVX). We conclude that sex differences in recovery of VT and phrenic output are present at 2 weeks post-C2HS. These differences are consistent with the hypothesis that ovarian sex hormones influence respiratory recovery after cervical spinal cord injury.

Brain metabolism of nutritionally essential polyunsaturated fatty acids depends on both the diet and the liver

Plasma α-linolenic acid ( α-LNA, 18:3n-3) and linoleic acid (LA, 18:2n-6) do not contribute significantly to the brain content of docosahexaenoic acid (DHA, 22:6n-3) or arachidonic acid (AA, 20:4n-6), respectively, and neither DHA nor AA can be synthesized de novo in vertebrate tissue. Therefore, measured rates of incorporation of circulating DHA and AA into brain exactly represent their rates of consumption by brain. Positron emission tomography (PET) has been used to show, based on this information, that the adult human brain consumes AA and DHA at rates of 17.8 and 4.6 mg/day, respectively, and that AA consumption does not change significantly with age. In unanesthetized adult rats fed an n-3 PUFA “adequate” diet containing 4.6% α-LNA (of total fatty acids) as its only n-3 PUFA, the rate of liver synthesis of DHA was more than sufficient to maintain brain DHA, whereas the brain's rate of DHA synthesis is very low and unable to do so. Reducing dietary α-LNA in the DHA-free diet led to upregulation of liver but not brain coefficients of α-LNA conversion to DHA and of liver expression of elongases and desaturases that catalyze this conversion. Concurrently, brain DHA loss slowed due to downregulation of several of its DHA-metabolizing enzymes. Dietary α-LNA deficiency also promoted accumulation of brain docosapentaenoic acid (22:5n-6), and upregulated expression of AA-metabolizing enzymes, including cytosolic and secretory phospholipases A 2 and cyclooxygenase-2. These changes, plus reduced levels of brain derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) in n-3 PUFA diet deficient rats, likely render their brain more vulnerable to neuropathological insults.

Regulation by diet and liver of brain metabolism of nutritionally essential polyunsaturated fatty acids

It is possible to inject radiolabeled polyunsaturated fatty acids (PUFAs) intravenously to quantify rates of brain and liver PUFA metabolism in the intact organism, in relation to diet, aging or disease. Because circulating α-linolenic acid (α-LNA, 18:3n-3) and linoleic acid (LA, 18:2n-6) in plasma do not contribute to brain docosahexaenoic acid (DHA, 22:6n-3) or arachidonic acid (AA, 20:4n-6), respectively, and DHA and AA cannot be synthesized de novo in vertebrate tissue, rates of incorporation of circulating DHA or AA into brain provide exact measurements of their rates of consumption by brain. Using positron emission tomography imaging, we reported that the adult human brain consumes AA and DHA at rates of 17.8 and 4.6 mg/day, respectively, and that the rate of AA consumption doesn’t change with age. In unanesthetized adult rats fed an n-3 PUFA “adequate” diet containing 4.6% (of total fatty acids) α-LNA as its only n-3 PUFA, the liver secretes DHA derived from circulating α-LNA ten-times faster than the brain consumes DHA; thus the liver is capable of supplying all the brain’s DHA. With a low dietary α-LNA level, rat liver coefficients of α-LNA conversion to DHA are increased because of increased liver elongase and desaturase activities, and DHA loss from brain is slowed due to downregulated DHA-metabolizing enzymes, including Ca<sup>2+<sup/>-independent phospholipase A<sub>2<sub/> (iPLA<sub>2<sub/>). The n-3 PUFA “deficient” diet also increases brain expression of AA-metabolizing enzymes, cytosolic cPLA<sub>2<sub/>, secretory sPLA<sub>2<sub/> and cyclooxygenase-2, and the brain docosapentaenoic acid (22:5n-6) concentration. These changes, plus reduced expression of brain derived neurotrophic factor (BDNF) caused by the “deficient” diet, likely increase brain vulnerability to excitotoxicity and inflammation.

Imaging Brain Phospholipase A2 Activation in Awake Rats in Response to the 5-HT2A/2C Agonist (±)2,5-Dimethoxy-4-Iodophenyl-2-Aminopropane (DOI)

Incorporation coefficients k(*) of intravenously injected [(3)H]arachidonic acid from blood into brain reflect the release from phospholipids of arachidonic acid by receptor-initiated activation of phospholipase A(2) (PLA(2)). In unanesthetized adult rats, 2.5 mg/kg intraperitoneally (i.p.) (+/-)2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), which is a 5-HT(2A/2C) receptor agonist, has been reported to produce the behavioral changes of what is known as the 5-HT(2) syndrome, but only a few small regional decrements in brain glucose metabolism. In this study, 2.5 mg/kg i.p. DOI, when administered to unanesthetized rats, produced widespread and significant increases, of the order of 60%, in k(*) for arachidonate, particularly in neocortical brain regions reported to have high densities of 5-HT(2A) receptors. The increases could be entirely blocked by chronic pretreatment with mianserin, a 5-HT(2) receptor antagonist. The results suggest that the 5-HT(2) syndrome involves widespread brain activation of PLA(2) via 5-HT(2A) receptors, leading to the release of the second messenger, arachidonic acid. Chronic mianserin, a 5-HT(2) antagonist, prevents this activation.

NO and the hypometabolic and hypothermic responses to hypoxia in the rat

Nitric oxide (NO) has been identified as a possible mediator of the thermoregulatory and respiratory responses to hypoxia. The present study was designed to assess the effects of an NO donor ( S-nitroso- N-acetylpenicillamine, SNAP) injected systemically (0.5–2.0 mg/kg) in unanesthetized adult rats studied at ambient temperatures (Ta) of 26, 24, and 15°C. The metabolic rate ( V ̇ O 2 ), ventilation ( V ̇ ), and colonic temperature (Tc) were recorded while the animals were exposed to normoxia or to hypoxia (FI O 2 =0.11). During normoxia, at all values of Ta, SNAP increased V ̇ O 2 and V ̇ and the decrease in Tc observed following saline injection was prevented or attenuated. However, SNAP did not modify the hypometabolic, hypothermic, and hyperventilatory responses to hypoxia. We concluded that, confirming previous studies, NO may play a role in the control of V ̇ O 2 and V ̇ in normoxia. The failure to observe any effect following the systemic NO donor injection during hypoxia may indicate opposing actions of the drug at different sites resulting in no net changes in ventilatory and metabolic responses to hypoxia.

Role of nitric oxide in hypoxic hypometabolism in rats.

Because it has been recently suggested that nitric oxide (NO) may mediate the effects of hypoxia on body temperature and ventilation, the present study was designed to assess more completely the effects of a neuronal NO synthase inhibitor (7-nitroindazole, 25 mg/kg ip), at ambient temperature of 26 and 15 degrees C, on the ventilatory (V), metabolic (O(2) consumption), and thermal changes (colonic and tail temperatures) induced by ambient hypoxia (fractional inspired O(2) of 11%) or CO hypoxia (fractional inspired CO of 0.07%) in intact, unanesthetized adult rats. At both ambient temperatures, 7-nitroindazole decreased oxygen consumption, colonic temperature, and V in normoxia. The drug reduced ambient or CO hypoxia-induced hypometabolism and ventilatory response, but the hypothermia persisted. It is concluded that NO arising from neural NO synthase plays an important role in the control of metabolism and V in normoxia. As well, it mediates, in part, the hypometabolic and the ventilatory response to hypoxia. The results are consistent with the notion that central nervous system hypoxia resets the thermoregulatory set point by decreasing brain NO.

Intravenous injection of [1‐14C]arachidonate to examine regional brain lipid metabolism in unanesthetized rats

We examined the metabolic disposition and brain distribution of an unsaturated fatty acid, [1-14C]arachidonate, between 5 and 240 min following its intravenous bolus injection in unanesthetized adult rats. Injected [1-14C]arachidonate was cleared rapidly from plasma, with less than 10% remaining by 2 min. Total brain radioactivity, 0.2% of the injected dose, was near maximal by 5 min, reached a peak by 15 min, then slowly declined. Radioactivity in brain lipids constituted greater than 82% of the total brain radioactivity at all times. Radioactivity in aqueous-soluble metabolites was greatest at 5 min (13% of total) and declined to 5% by 240 min. Protein pellet-associated radioactivity gradually rose to a peak of 7% by 120 min. Within the lipid fraction, more than 92% of radioactivity was in glycerolipids, with greater than 81% in phospholipids. Radioactivity in inositol phosphoglyceride was maximal at 5 min (47% of phospholipid radioactivity); and declined to 34% by 20 min, whereas radioactivity in choline phosphoglyceride peaked at 15 min (41% of phospholipid radioactivity) and was constant thereafter. In contrast, radioactivity in ethanolamine phosphoglycerides increased from 7 to 17% during the course of the experiment. Quantitative autoradiography of brain sections indicated incorporation of [1-14C]arachidonate into gray-matter regions was 1.5- to threefold that into white-matter regions. The data were analyzed in terms of a model for brain fatty acid uptake from plasma. Estimates of unidirectional transfer constants, k, for [1-14]arachidonate from plasma to brain regions with an intact blood-brain barrier ranged from 0.0005 to 0.0015 ml.sec-1.g-1 and were correlated with those for [9,10-3H]palmitate. The results indicate that brain phospholipid metabolism in awake animals can be examined regionally and quantitatively using intravenous injection of [1-14C]-arachidonate combined with quantitative autoradiography and biochemical analysis.