Rats Breathing Room Air Research Articles

Extended oxygen supplementation after thoracotomy in rats may improve welfare.

A retrospective comparison of welfare indicators in male rats undergoing thoracotomy for intrapleural dosing is presented. The initial cohort (n = 7) breathed room air after recovery from anaesthesia, while later cohorts (n = 12) had oxygen supplementation for up to 48 h post-surgery. Rats breathing room air sustained a statistically significant average body-weight loss of -1.62% (±1.7%) 2 days after surgery, compared with rats given oxygen supplementation, which maintained a mean weight gain of 0.87% (±1.75%) (p = 0.009). Oxygen-supplemented rats also had lower pain scores on the evening after surgery (median 0.075 [range 0-1.75] vs. median 1.5 [range 0.5-2]). This difference was not statistically significant (p = 0.063) but may be of clinical significance. All rats displayed transient sedation after post-operative opioid administration on the day of surgery, and hypoxaemia (SpO2 <90%) was observed in rats maintained on room air. Use of an oxygen concentrator to provide post-operative extended oxygen supplementation was easy to implement and may improve animal welfare post-thoracotomy.

Chronic Hyperoxia Augments the Hypercapnic Ventilatory Response in Adult Rats

Rats exposed to 4 – 14 days of 60% O2 as adults exhibit a modest enhancement of their hypoxic ventilatory response (HVR) (Danielson &amp; Bavis, Physiology 38(S1): 5730119, 2023). To determine whether this plasticity is unique to the HVR or a general increase in excitability of the respiratory control system, we tested the hypothesis that chronic hyperoxia would also enhance the hypercapnic ventilatory response (HCVR). The ventilatory response to 7% CO2 was measured by whole-body plethysmography in adult, Sprague-Dawley rats immediately after 4 and 14 days of exposure to 60% O2 or an equivalent time period in room air. Baseline ventilation and the HCVR were unchanged after 4 days of hyperoxia. The HCVR was increased after 14 days (Treatment × FICO2, P=0.004), with hypercapnic ventilation being approximately 15% greater than in control rats. However, this effect was no longer significant after accounting for variation in metabolic rate, and the HCVR was fully recovered when rats were restudied four weeks after they were returned to room air. In a follow-up experiment, pulse oximetry was performed in isoflurane-anesthetized rats breathing room air or 12% O2. Exposure to 4 – 14 days of 60% O2 did not impair gas exchange, so augmented ventilatory responses likely reflect changes in the neural control of breathing and/or metabolism rather than differences in the levels of hypoxemia and hypercapnia experienced by rats during the tests. In conclusion, chronic hyperoxia elicits plasticity in both the HVR and HCVR of adult rats, but these responses recover in less than one month after rats are returned to room air. Supported by NIH grant P20 GM-103423 (Maine INBRE). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of hydrogen inhalation on cardiovascular and interstitial components of pulmonary hypertension in rats

Hydrogen is known to have selective antioxidant properties. It binds highly reactive hydroxyl radicals. The pathogenesis of the monocrotaline animal model of pulmonary hypertension is associated with oxidative stress and leads to all the symptoms of pulmonary hypertension (PH) and interstitial lung disease (ILD) associated with hypertension.The aim of this work was to study the effect of 4% hydrogen inhalations on the symptoms of PH and ILD in rats.Methods. To model monocrotaline-induced pulmonary hypertension (MCT-PH), two groups of animals received a single subcutaneous injection of monocrotaline (MCT) on day 1. The control group was injected subcutaneously with MCT solvent only. The animals receiving MCT were further divided into 2 subgroups. Subgroup 1 rats breathed room air and subgroup 2 rats breathed a mixture of room air and 4% hydrogen. The regular inhalations continued until day 21. On day 21, hemodynamic parameters were measured under urethane anesthesia, the heart and its components and the lungs were weighed, and lung tissue was preserved for morphological study.Results. The inhalation had no effect on the main cardiovascular symptoms of PH, but a positive effect on the state of the connective tissue of the lungs affected by PH was shown. The mast cell response was reduced both quantitatively and functionally. There was a decrease in tryptase expression by mast cells, with predominance of the forms without signs of degranulation. TGF-β secretion was also significantly reduced and visualized by immunopositive cells in alveolar cellular structures and vessel walls.Conclusion. Inhalation of 4% hydrogen reduces inflammation and fibrosis of lung tissue during the development of MCT-PH.

Evaluation of Betulinic Acid Derivatives as PET Tracers for Hypoxia-Induced Carbonic Anhydrase IX (CA IX) Expression.

Non-invasive visualisation of the expression of hypoxia-related proteins, such as carbonic anhydrase IX (CA IX), by positron emission tomography (PET) could provide important information on the oxygenation status of tumours. Since betulinic acid derivatives bind specifically to CA IX the aim of the study was the development betulinic acid-based 68Ga-labelled PET tracers and to evaluate the hypoxia detecting properties in vitro and in vivo. The binding of betulinic acid (B-DOTA) and betulinyl-3-sulfamate (BS-DOTA) was assessed in two rat tumour cell lines (AT1 prostate and Walker-256 mammary carcinomas). AT1 cells express CA IX in a hypoxia-dependent manner whereas Walker-256 cells, expressing almost no CA IX in wildtype, were transfected with the rat Car9 gene. In vivo measurements were carried out in a small animal PET/CT in AT1 tumours in rats breathing room air, 8% or 100% O2. In AT1 cells hypoxia-induced overexpression of CA IX led to a stronger binding of BS-DOTA but not of B-DOTA. The BS-DOTA binding correlated linearly with the CA IX protein expression and could be blocked by an excess of unlabelled tracer. In the transfected Walker-256 cells no specific binding of either of the tracers was seen. In vivo the intratumoral accumulation of BS-DOTA was increased in animals kept under inspiratory hypoxia and reduced by hyperoxia. Therefore, betulinyl-3-sulfamate could be used as a PET tracer of CA IX expression in tumours and to provide information about the oxygenation status. However, accumulation data indicated that binding not only depends on hypoxia-induce CA IX expression but also on the tumour-line-specific basal expression and on the initial oxygenation status of the tumour.

Selective optogenetic stimulation of RTN neurons drives ventilation and active expiration in unanesthetized rats

BackgroundThe retrotrapezoid nucleus (RTN) consists of neurons that are chemosensitive (i.e., CO2/pH sensitive), glutamatergic, Phox2b+ and Neuromedin B+ (Nmb+). Lentiviral vectors currently used to transduce RTN neurons with various opto‐ or pharmacogenetic actuators contain a Phox2b‐activated promoter, PRSx8. When injected into the RTN, these vectors also transduce substantial numbers of nearby C1 and catecholaminergic neurons. The purpose of the present study was to determine the effects on breathing elicited by selectively activating RTN neurons.MethodsTo selectively transduce RTN neurons we microinjected a mixture of Lenti‐PRSx8‐ChR2‐mCherry and AAV5‐FLEX‐taCasP3‐TEVp into the RTN of adult TH‐Cre rats. One month after the surgery, RTN neurons were stimulated with blue light (473 nm, 10 ms pulse, 20 Hz, for 20 s). All the measurements were conducted inside a plethysmography chamber. Diaphragm (DIA) and abdominal (ABD) muscle activity were recorded in conscious conditions. Results (mean ± SD), were analyzed using one‐ or two‐way ANOVA as required and differences were considered significant at P&lt;0.05.ResultsRTN neurons (Nmb+, TH−) were selectively transduced (96.8 % ± 4) with the PRSx8 vector, the FLEX‐caspase AAV5 having caused the apoptotic death of C1 neurons (TH+) in the area where neurons were transduced with ChR2. Selective stimulation of RTN (NMB+ neurons; n=6) increased respiratory rate during wake and NREM sleep but not during REM sleep (Δ 30 ± 7 vs. 22.7 ± 6 vs. 4 ± 4 bpm from baseline), increased tidal volume also during quiet waking and non‐REM but not REM sleep (Δ 0.15 ± 0.07 vs. 0.13 ± 0.04 vs. 0.02 ± 0.007 mL/100g) and VE also during wake and non‐REM sleep but not REM (27.8 ± 11 vs. 21.4 ± 8 vs. 3.1 ±1 mL/100g/min). Selective RTN stimulation in rats breathing room air elicited active expiration (from 8.4 ± 7 to 15.9 ± 8 % of normalized abdominal EMG recorded at 9% FiCO2, n=3). RTN stimulation produced greater active expiration in rats exposed to 6% FiCO2 (from 56.3 ± 32 to 125.3 ± 104 % of normalized abdominal EMG).ConclusionsSelective RTN neuron stimulation drives breathing amplitude and frequency during wake, NREM but not in REM sleep, and elicits active expiration, especially at elevated levels of FiCO2.Support or Funding InformationSupported by grants from the National Institutes of Health (HL28785, 18 HL074011) and the Congenital Central Hypoventilation Syndrome Foundation to PGG.

Blood Pressure Regulation by the Rostral Ventrolateral Medulla in Conscious Rats: Effects of Hypoxia, Hypercapnia, Baroreceptor Denervation, and Anesthesia.

Current understanding of the contribution of C1 neurons to blood pressure (BP) regulation derives predominantly from experiments performed in anesthetized animals or reduced ex vivo preparations. Here, we use ArchaerhodopsinT3.0 (ArchT) loss-of-function optogenetics to explore BP regulation by C1 neurons in intact, unanesthetized rats. Using a lentivirus that expresses ArchT under the Phox2b-activated promoter PRSx8 (PRSx8-ArchT), ∼65% of transduced neurons were C1 (balance retrotrapezoid nucleus, RTN). Other rats received CaMKII-ArchT3.0 AAV2 (CaMKII-ArchT), which transduced C1 neurons and larger numbers of unidentified glutamatergic and GABAergic cells. Under anesthesia, ArchT photoactivation reduced sympathetic nerve activity and BP and silenced/strongly inhibited most (7/12) putative C1 neurons. In unanesthetized PRSx8-ArchT-treated rats breathing room air, bilateral ArchT photoactivation caused a very small BP reduction that was only slightly larger under hypercapnia (6% FiCO2), but was greatly enhanced during hypoxia (10 and 12% FiO2), after sino-aortic denervation, or during isoflurane anesthesia. The degree of hypotension correlated with percentage of ArchT-transduced C1 neurons. ArchT photoactivation produced similar BP changes in CaMKII-ArchT-treated rats. Photoactivation in PRSX8-ArchT rats reduced breathing frequency (FR), whereas FR increased in CaMKII-ArchT rats. We conclude that the BP drop elicited by ArchT activation resulted from C1 neuron inhibition and was unrelated to breathing changes. C1 neurons have low activity under normoxia, but their activation is important to BP stability during hypoxia or anesthesia and contributes greatly to the hypertension caused by baroreceptor deafferentation. Finally, C1 neurons are marginally activated by hypercapnia and the large breathing stimulation caused by this stimulus has very little impact on resting BP.SIGNIFICANCE STATEMENT C1 neurons are glutamatergic/peptidergic/catecholaminergic neurons located in the medulla oblongata, which may operate as a switchboard for differential, behavior-appropriate activation of selected sympathetic efferents. Based largely on experimentation in anesthetized or reduced preparations, a rostrally located subset of C1 neurons may contribute to both BP stabilization and dysregulation (hypertension). Here, we used Archaerhodopsin-based loss-of-function optogenetics to explore the contribution of these neurons to BP in conscious rats. The results suggest that C1 neurons contribute little to resting BP under normoxia or hypercapnia, C1 neuron discharge is restrained continuously by arterial baroreceptors, and C1 neuron activation is critical to stabilize BP under hypoxia or anesthesia. This optogenetic approach could also be useful to explore the role of C1 neurons during specific behaviors or in hypertensive models.

Inactivity‐Induced Phrenic Motor Facilitation underlies Spontaneous Recovery of Ventilation and Diaphragm EMG activity Following Cervical Spinal Injury

Cervical spinal injury disrupts neural pathways to phrenic motor neurons, thereby causing diaphragm paresis and paralysis and diminished breathing capacity. Some spontaneous functional recovery in the capacity to generate phrenic nerve or diaphragm activity is observed days‐to‐months post‐injury via mechanisms that not understood. We have previously found that prolonged reductions in spinal synaptic inputs to phrenic motor neurons in uninjured rats results in a PKCz‐dependent, rebound enhancement of phrenic burst amplitude, a form of spinal plasticity known as inactivity‐induced phrenic motor facilitation (iPMF). Little is known regarding the role for iPMF in the control of breathing. Here, we tested the hypothesis that mechanisms giving rise to iPMF underlie spontaneous recovery of ventilation and diaphragm EMG activity following a high cervical spinal injury. Sprague Dawley rats (Harlan colony 217, 12–16 weeks old) chronically instrumented with radiotelemetry for bilateral diaphragm electromyogram (EMG) recordings received small interfering RNAs directed against PKCz or non‐targeting siRNAs in the interpleural space (100pg/side) three days prior to a C3 midline contusion. Three days following contusion, EMG and plethysmography were recorded in unanesthetized rats breathing room air (FIO2/FICO2=0.21/0.0, NX) or with maximal chemoreceptor stimulation (FIO2/FICO2= 0.105/0.07, MCS). In injured rats receiving control injections, tidal volume under both inspired gas concentrations and diaphragm EMG during MCS showed minimal change from baseline conditions (TV NX: 99%, TV MCS: 90%, diaphragm EMG right: 95%, left: 100%). By contrast, in injured rats receiving siPKCz, tidal volume during normoxia and MCS (63% NX, 90% MCS) and electrical activity of the diaphragm during MCS (right: 85%, left: 80%) were reduced from pre‐injury levels, suggesting that impairment of iPMF prevented spontaneous recovery of phrenic motor output (and hence, diaphragm EMG activity) following spinal injury. Thus, we speculate that iPMF is a form of compensatory plasticity that senses and responds to reductions in spinal synaptic inputs to phrenic motor neurons to maintain stable phrenic motor output following spinal injury.Support or Funding InformationSupported by NIH R01‐HL105511, DoD SC120226 and NIH T32‐OD010423‐09

Daily acute intermittent hypoxia elicits functional recovery of diaphragm and inspiratory intercostal muscle activity after acute cervical spinal injury

A major cause of mortality after spinal cord injury is respiratory failure. In normal rats, acute intermittent hypoxia (AIH) induces respiratory motor plasticity, expressed as diaphragm (Dia) and second external intercostal (T2 EIC) long-term facilitation (LTF). Dia (not T2 EIC) LTF is enhanced by systemic adenosine 2A (A2A) receptor inhibition in normal rats. We investigated the respective contributions of Dia and T2 EIC to daily AIH-induced functional recovery of breathing capacity with/without A2A receptor antagonist (KW6002, i.p.) following C2 hemisection (C2HS). Rats received daily AIH (dAIH: 10, 5-min episodes, 10.5% O2; 5-min normoxic intervals; 7 successive days beginning 7days post-C2HS) or daily normoxia (dNx) with/without KW6002, followed by weekly (reminder) presentations for 8weeks. Ventilation and EMGs from bilateral diaphragm and T2 EIC muscles were measured with room air breathing (21% O2) and maximum chemoreceptor stimulation (MCS: 7% CO2, 10.5% O2). dAIH increased tidal volume (VT) in C2HS rats breathing room air (dAIH+vehicle: 0.47±0.02, dNx+vehicle: 0.40±0.01ml/100g; p<0.05) and MCS (dAIH+vehicle: 0.83±0.01, dNx+vehicle: 0.73±0.01ml/100g; p<0.001); KW6002 had no significant effect. dAIH enhanced contralateral (uninjured) diaphragm EMG activity, an effect attenuated by KW6002, during room air breathing and MCS (p<0.05). Although dAIH enhanced contralateral T2 EIC EMG activity during room air breathing, KW6002 had no effect. dAIH had no statistically significant effects on diaphragm or T2 EIC EMG activity ipsilateral to injury. Thus, two weeks post-C2HS: 1) dAIH enhances breathing capacity by effects on contralateral diaphragm and T2 EIC activity; and 2) dAIH-induced recovery is A2A dependent in diaphragm, but not T2 EIC. Daily AIH may be a useful in promoting functional recovery of breathing capacity after cervical spinal injury, but A2A receptor antagonists (e.g. caffeine) may undermine its effectiveness shortly after injury.

The Effect of Chronic Long-Term Intermittent Hypobaric Hypoxia on Bone Mineral Density in Rats: Role of Nitric Oxide

Intermittent hypoxia is the most common pattern of hypoxic exposure in humans. The effect of chronic long-term intermittent hypobaric hypoxia (CLTIHH) on bone metabolism is not investigated. We examined the effect of CLTIHH on bone metabolism and the role of nitric oxide (NO) in this process. The rats were divided into three groups in this study. The animals in groups I and II have been exposed to CLTIHH. The animals in group II were also treated with nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester. To obtain CLTIHH, rats were placed in a hypobaric chamber (430 mm Hg; 5 h/day, 5 days/week, 5 weeks). The group III (control) rats breathed room air in the same environment. At the begining of the experiments, bone mineral density (BMD) of the animals were measured, and blood samples were collected from the tail vein. After the 5-week CLTIHH period, the same measurements were repeated. Parathyroid hormone, calcium, phosphate, bone alkaline phosphatase (b-ALP), NO, interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha levels were determined. The cytokines, NO levels, and BMD in CLTIHH-induced rats were higher compared with baseline and control values. The cytokines, b-ALP, and BMD increased while NO levels decreased in the group II compared with baseline values. BMD values of group II were lower than group I but higher than control group. Our results suggested that CLTIHH has positive effects on bone density. Intermittent hypoxia protocols may be developed for treatment and prevention of osteopenia and osteoporosis.

Hypothermic protection in an acute hypoxia model in rats: Acid–base and oxidant/antioxidant profiles

Aim of the study Recent works demonstrate the benefits of hypothermia when used to preserve brain, cardiac, hepatic, and intestinal function against hypoxic-ischemic injury. However, it is also known that hypothermia affects systemic parameters and also induces the generation of reactive oxygen species in cells and tissues. Here we studied the acid–base related parameters and the antioxidant–oxidant effects of deep hypothermia induction before an acute hypoxic insult in rats. Methods Acid–base indicators and parameters related to oxidative stress were analyzed in hypothermic rats (21–22 °C) breathing room air during 2 h (control hypothermia), and hypothermic animals switched to hypoxic air (10% O 2) during the second hour (hypothermia hypoxia group), and they were compared with corresponding normothermia groups maintained at 37 °C (control normothermia and normothermia hypoxia groups). Results Mild metabolic acidosis appeared early in arterial blood during hypothermia. After exposure to hypoxia, evidence of tissue injury (plasma transaminases and blood lactate) and oxidative stress (increase in lipid peroxidation, decrease in glutathione levels and in the glutathione reduction potential in liver) was found. In contrast, in the hypothermia hypoxia group, plasmatic parameters remained as the control values, and the hepatic glutathione reduction potential were significantly more negative when compared with the normothermia hypoxia group. Conclusions We propose that acidosis induced by hypothermia contributes to the maintenance of intracellular reduction potential in liver, regarding the GSSG/2GSH couple and may help to increase plasmatic antioxidant pool. Our findings provide new insights into the protective effects of hypothermia in vivo.

Increased sympathetic activity in rats submitted to chronic intermittent hypoxia (CIH) is coupled to enhanced late expiratory activity

The mechanisms triggering the sympathetic overactivity in CIH rats are not understood. In the present study, we explored whether juvenile rats exposed to CIH exhibit changes in central respiratory‐sympathetic coupling. CIH was induced with 6% O2 for 40 s every 9 min, 8 h/day for 10 days. On the 11th day, awake CIH rats breathing room air showed a higher mean arterial pressure than controls (101 ± 3 vs 89 ± 3 mmHg, n = 14). Recordings of phrenic, thoracic sympathetic (tSNA), cervical vagus (cVN) and abdominal nerve (Abd) activities were made in the working heart‐brainstem preparation. During late expiration (Late E), tSNA was greater in CIH (n = 9) than in control rats (52 ± 5 vs 40 ± 3%, n = 11; P &lt; 0.05). Moreover, CIH rats (n = 6), but not controls (n = 5), also exhibited a peak of activity in Abd during Late E with reduced post‐inspiratory activity in cVN. These observations suggest that CIH alters the breathing pattern evoking Late E activity in the Abd. We propose that this Late E discharge in CIH rats contributes with an additional excitatory synaptic drive to the sympathetic outflow and, in turn, raises arterial pressure. Further studies are required to identify the neuronal populations and the correspondent neurochemical mechanisms responsible for the altered respiratory‐sympathetic entrainment in CIH rats. Supported by CNPq and FAPESP (Brazil) and BHF (UK).

Blocking ET-1 Receptors Does Not Correct Subnormal Retinal Oxygenation Response in Experimental Diabetic Retinopathy

To test the hypothesis that bosentan (a dual ET(A)/ET(B) receptor antagonist) corrects a subnormal retinal oxygenation response in the STZ-induced diabetic rat. In benchtop experiments, ET-1 was acutely injected into the vitreous of control and 5- to 7-day bosentan-treated nondiabetic rats. Major retinal vessel diameters were analyzed from ADPase-stained flatmounts. Retinal oxygenation (DeltaPo(2)), an established early surrogate marker of drug treatment efficacy, was measured by MRI during a 2-minute carbogen inhalation challenge in four groups: control rats (n = 7), control rats treated with bosentan (n = 7), 3-month diabetic rats (n = 9), and 3-month diabetic rats treated with bosentan (n = 5). Effect of baseline differences was studied in control rats breathing either room air (n = 5) or 12% oxygen breathing (n = 5) before a 2-minute carbogen provocation. ET-1 produced a significant (P < 0.05) reduction in retinal arterial diameter that was suppressed (P > 0.05) in rats fed bosentan chow admix. For all groups, no MRI baseline signal intensity differences were found (P > 0.05). Also, comparisons between baseline room air and 12% conditions and control rats fed normal chow or a bosentan admix both produced similar (P > 0.05) panretinal DeltaPo(2). In treated and untreated diabetes groups, inferior hemiretinal DeltaPo(2) remained normal (P > 0.05), but superior hemiretinal DeltaPo(2) was subnormal (P < 0.05). Because subnormal retinal DeltaPo(2) after drug treatment is a biomarker of subsequent vascular histopathology, the present data raise the possibility that retinal ET-1 does not play a key role in the pathogenesis of diabetic retinopathy.

Cerebral Blood Flow and Brain Oxygenation in Rats Breathing Oxygen under Pressure

Hyperbaric oxygen (HBO(2)) increases oxygen tension (PO(2)) in blood but reduces blood flow by means of O(2)-induced vasoconstriction. Here we report the first quantitative evaluation of these opposing effects on tissue PO(2) in brain, using anesthetized rats exposed to HBO(2) at 2 to 6 atmospheres absolute (ATA). We assessed the contribution of regional cerebral blood flow (rCBF) to brain PO(2) as inspired PO(2) (PiO(2)) exceeds 1 ATA. We measured rCBF and local PO(2) simultaneously in striatum using collocated platinum electrodes. Cerebral blood flow was computed from H(2) clearance curves in vivo and PO(2) from electrodes calibrated in vitro, before and after insertion. Arterial PCO(2) was controlled, and body temperature, blood pressure, and EEG were monitored. Scatter plots of rCBF versus PO(2) were nonlinear (R(2)=0.75) for rats breathing room air but nearly linear (R(2)=0.88-0.91) for O(2) at 2 to 6 ATA. The contribution of rCBF to brain PO(2) was estimated at constant inspired PO(2), by increasing rCBF with acetazolamide (AZA) or decreasing it with N-nitro-L-arginine methyl ester (L-NAME). At basal rCBF (78 mL/100 g min), local PO(2) increased 7- to 33-fold at 2 to 6 ATA, compared with room air. A doubling of rCBF increased striatal PO(2) not quite two-fold in rats breathing room air but 13- to 64-fold in those breathing HBO(2) at 2 to 6 ATA. These findings support our hypothesis that HBO(2) increases PO(2) in brain in direct proportion to rCBF.

Serotonin 1A Receptor Agonists Reverse Respiratory Abnormalities in Spinal Cord-Injured Rats

Contusion spinal cord injury (SCI) at T8 produces respiratory abnormalities in conscious rats breathing room air and challenged with CO2. In seeking ways to improve respiration after SCI, we tested drugs that stimulate serotonin 1A (5-HT1A) receptors, based on our previous findings that these agents can counteract respiratory depression produced by morphine overdose. Respiratory function was measured with a head-out plethysmograph system in conscious rats. T8 SCI rats (n = 5) showed decreased tidal volume (Vt; 0.90 +/- 0.02-0.66 +/- 0.03 ml; p < 0.05) and increased respiratory rate (f;91 +/- 3.7-132 +/- 5.7 breaths/min; p < 0.05) with room air ventilation at 24 hr after injury. They also exhibited a diminished response to the respiratory stimulating effect of 7% CO2; minute ventilation increased to 250 +/- 17 ml/min before, but only to 162 +/- 15 ml/min at 24 hr after SCI (p < 0.05). Respiratory deficits during room air ventilation were also observed at 7 d after injury (n = 3). Treatment with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylmino)tetralin (8-OH-DPAT; 250 microg/kg, i.p.) at 24 hr (n = 5) or 7 d (n = 3) after injury normalized Vt, f, and the respiratory response to 7% CO2. Identical results were obtained with another 5-HT1A receptor agonist, buspirone (1.5 mg/kg, i.p.; n = 3). In contrast, intraperitoneal saline vehicle administration (n = 5) showed no beneficial effects on SCI-impaired respiration. Finally, pretreatment with a specific antagonist of 5-HT1A receptors, 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide (3 mg/kg, i.p.; n = 3) given 20 min before 8-OH-DPAT, prevented 8-OH-DPAT from restoring respiration to normal. Our results demonstrate that drugs that stimulate 5-HT1A receptors counteract respiratory abnormalities in conscious rats after SCI.

Correlation of a simple direct measurement of muscle pO 2 to a clinical ischemia index and histology in a rat model of chronic severe hindlimb ischemia

Purpose: The lack of suitable experimental models of chronic severe limb ischemia and deficiencies in the available methods that allow for direct intermittent measurement of regional limb perfusion are obstacles to the evaluation of recently developed molecular strategies to reverse severe limb ischemia. Our aim was to develop a model of clinically relevant severe limb ischemia and correlate a simple direct measurement of muscle pO 2 to a clinical ischemia index, muscle mass, and capillary density. Methods: Severe hindlimb ischemia was induced in 44 adult rats with ligation of the left common iliac artery, the femoral artery, and their branches. The effect of ischemia on muscle pO 2 was measured in the left gastrocnemius with room air and with 100% oxygen at 3, 10, 24, and 40 days after ischemia was induced. Clinical ischemia index, muscle mass, cellular proliferation, and capillary density also were assessed. Results: The clinical ischemia index of the left limb was most severe at day 10, with evidence of pressure sores, a pale and dusky limb, and abnormal gait. With the rats breathing room air, muscle pO 2 was significantly lower in the left limbs than in the right limbs at days 3, 10, 24, and 40. After an oxygen challenge (100% O 2), muscle pO 2 was significantly lower at 3, 10, and 40 days. At 3 days, the fraction of muscle mass per total body weight of the left tibialis anterior (TA) was significantly greater than the right TA as a result of edema and inflammation. By days 10, 24, and 40, the left gastrocnemius and TA masses were significantly less than the right as a result of muscle atrophy. Histopathology showed severe necrosis in the left gastrocnemius and TA on day 3. Inflammation was greatest by day 10. Necrotic muscle regenerated but remained atrophic at 40 days. The TA was slower to recover than the gastrocnemius. Capillary densities and capillary-to-muscle fiber ratios were greater in the ischemic limb than in the normal limb at day 24. Cellular proliferation as determined with bromodeoxyuridine labeling reagent staining was maximal in the ischemic limb at day 3. Conclusion: We have developed a rat model of chronic severe hindlimb ischemia with persistent ischemia as shown with a simple direct measurement of muscle pO 2 for up to 40 days. This model of severe hindlimb ischemia may be applicable for future studies of molecular strategies to treat severe limb ischemia in humans. (J Vasc Surg 2002;36:172-9.)

A Bayesian Model for Detecting Acute Change in Nonlinear Profiles

We propose a model for longitudinal data with random effects that includes model-based smoothing of measurements over time. This research is motivated by experiments evaluating the hemodynamic effects of various agents in tumor-bearing rats. In one set of experiments, the rats breathed room air, followed by carbogen (a mixture of pure oxygen and carbon dioxide). The experimental responses are longitudinal measurements of oxygen pressure measured in tissue, tumor blood flow, and mean arterial pressure. The nature of the recorded responses does not allow any meaningful parametric form to model these profiles over time. Additionally, response patterns differ widely across individuals. Therefore, we propose a nonparametric regression to model the profile data over time. We propose a dynamic state-space model to smooth the data at the profile level. Using the state parameters, we formally define “change” in the measured responses. A hierarchical extension allows inference to include a regression on covariates. The proposed approach provides a modeling framework for any longitudinal data, where no parsimonious parametric model is available at the level of the repeated measurements and a hierarchical modeling of some feature of a smooth fit for these profiles data is desired. The proposed MCMC algorithm for inference on the hierarchical extension is appropriate in any hierarchical model in which posterior simulation for the submodels is significantly easier.

Behavioral and Anatomical Correlates of Chronic Episodic Hypoxia during Sleep in the Rat

The role played by chronic episodic hypoxia (EHYP) in the neurocognitive morbidity of obstructive sleep apnea (OSA) is unknown. Sleep recordings, Morris water maze experiments, and immunohistochemistry for NMDA NR1 glutamate receptor, c-fos protein, and apoptosis [nuclear immunoreactivity for single-stranded DNA and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assay] were conducted in EHYP-exposed Sprague Dawley male rats. Exposures consisted of up to14 d in an environmental chamber in which O(2) concentrations were cycled between 10 and 21% every 90 sec or 30 min during 12 hr of daylight. For the remaining 12 hr, EHYP rats breathed room air, while controls spent 14 d in room air. Although EHYP induced significant disruption of sleep architecture during the initial day of exposure, sleep patterns normalized thereafter. Marked increases in apoptosis occurred in the CA1 hippocampal region (sevenfold) and cortex (Cx; eightfold) after 1-2 d of EHYP but not in CA3 and were followed by decreases toward normoxic levels by 14 d. Double labeling for NMDA NR1 and c-fos revealed marked architectural disorganization in CA1 and Cx with increases in c-fos over time. Rats exposed to EHYP displayed significantly longer escape latencies and swim path lengths to escape a hidden platform during 12 training trials given over 2 d. Differences in the performances of EHYP and control rats, although reduced, persisted after 14 d of recovery. We conclude that EHYP is associated with marked cellular changes over time within neural regions associated with cognitive functions. Furthermore, EHYP impaired performance during acquisition of a cognitive spatial task without affecting sensorimotor function. Such changes may underlie components of the learning and memory impairments found in OSA.

Role of endothelin in intermittent hypoxia-induced hypertension.

Clinical studies suggest that sleep apnea causes systemic hypertension. In addition, patients with sleep apnea have elevated plasma levels of endothelin-1 (ET-1). We hypothesized that the intermittent hypoxia/hypercapnia (IH) associated with sleep apnea causes hypertension by increasing ET-1 production. To test this hypothesis, rats with arterial and venous catheters were placed in Plexiglas chambers. IH rat chambers were flushed with an N(2)-CO(2) mixture for 90 seconds to achieve hypoxia/hypercapnia (5% O(2)-5% CO(2)) followed by 90 seconds of compressed air to achieve normoxia (21% O(2)-0% CO(2)). Control rat chambers were flushed with 90 seconds of air-air cycles. Cycles for both groups were repeated 8 hours per day for 11 days. Resting mean arterial pressure (MAP) and heart rate were recorded daily before the start of exposure. After 11 days, MAP was significantly elevated in IH rats compared with initial MAP (109+/-5 mm Hg initial, 139+/-11 mm Hg day 11) and compared with air-air rats (110+/-4 mm Hg). On day 11, cumulative doses of PD145065 (a nonselective ET-receptor antagonist) were administered intravenously to the rats breathing room air. PD145065 caused a dose-dependent decrease in MAP in IH rats but did not alter MAP in air-air rats. Plasma ET-1 measured by radioimmunoassay was significantly increased on days 5 and 11 in the IH rats compared with day 1 and compared with air-air rats. There was no significant change in plasma ET-1 over time in air-air rats. We conclude that IH exposure increases both MAP and plasma ET-1 and that the increased ET-1 may contribute to the hypertension.

Reproducibility of Scanning Laser Doppler Flowmetry in the Rat Retina and Optic Nervehead

Purpose: The purpose of this study is to confirm the reliability of scanning laser Doppler flowmetry in the rat retina and optic nervehead, and the validity of measuring changes of retinal blood flow in rats while breathing 100% oxygen. Methods: We used a commercially available scanning laser Doppler flowmeter. To ascertain reliability, five consecutive and separate perfusion measurements of 12 eyes of 12 anesthetized pigmented rats were performed. To evaluate the validity of the system, repeated measurements were taken in anesthetized rats breathing room air or 100% oxygen. This series of measurements was repeated three times. Results: The reliability coefficients of volume, flow, and velocity in the optic nervehead and the retina ranged from 0.80 to 0.83 and 0.77 to 0.82, respectively. After the first exposure to oxygen, the measured values of volume, flow, and velocity were reduced by an average of 20.9–24.0%, 21.2–28.2%, and 19.5–24.5%, respectively. After the values returned to the basal condition, the second and third exposures to oxygen yielded measured values that were reduced by the same amounts as at the first exposure. Conclusions: Scanning laser Doppler flowmetry provided relatively good reliability in measurements of blood flow in the rat retina and optic nervehead. This study has indicated the possibility of applying this system to the rat retina.

Subcutaneous tissue oxygen and carbon dioxide tensions during hyperbaric oxygenation: an experimental study in rats.

To investigate the response of subcutaneous tissue oxygen (O2) and carbon dioxide (CO2) tensions to hyperbaric oxygenation. Experimental study. University hospital, Finland. 10 Wistar rats. Subcutaneous tissue PO2 and PCO2 were directly measured with an implanted Silastic tube tonometer and capillary sampling technique while breathing air and exposed to hyperbaric oxygen (HBO) at 2.5 or 2.8 ATA pressure. Hyperbaric exposures were carried out in a large multiplace chamber pressurised with air. Subcutaneous tissue PO2 and PCO2. The mean subcutaneous PO2 rose from the baseline of 8 kPa (60 mmHg) to 16 kPa (112 mmHg) when rats breathed room air during pressurisation to 2.8 atm. When the rats breathed oxygen at 2.5 ATA the maximal mean tissue PO2 was four times higher than the mean starting value. During the HBO treatment at 2.8 ATA the tissue PO2 rose to a value about five times above baseline. The tissue PCO2 values almost doubled during the exposure to HBO at 2.5 ATA, probably because elimination of carbon dioxide was impaired. Measurements of tissue PO2 and PCO2 with an implanted Silastic tonometer and a capillary sampling technique can successfully be adapted to hyperbaric conditions. The method yielded reproducible results and is applicable to clinical use in hyperbaric medicine.