Why does opioids cause respiratory depression

Overall, we provide new evidence linking specific electrocortical changes to the severity of respiratory depression by opioids, which highlights the importance of considering the cortical and subcortical effects of opioids in addition to their impacts on breathing when evaluating opioid-induced respiratory depression.

Opioid drugs are widely used to alleviate pain but their effective use is limited by the life-threatening side-effect of respiratory depression 1. Opioids induce profound respiratory changes, such as sleep apnea and hypoventilation 2 , which can lead to complete respiratory arrest with opioid overdose. These drugs depress breathing by acting on key-structures of the brainstem 8 , which include the ventrolateral medulla 9 , the pons 10 , the rostral ventromedial medulla 7 , and the medullary raphe nuclei Inhibition of these respiratory structures and circuits directly induce respiratory depression by reducing respiratory rate 9 and respiratory chemosensitivity In addition to the direct effects of opioid drugs on such respiratory circuits, descending pathways originating from sub-cortical and cortical regions that modulate brainstem circuits are also inhibited by opioid drugs In fact, in addition to their analgesic and respiratory properties, opioid drugs also cause a profound sedative state 13 , reminiscent of sedation induced by other medications such as sedatives and anesthetics Similarly, low doses of morphine induced EEG changes consistent with fentanyl, i.

Although distinct from the EEG changes elicited by sleep, the EEG features associated with opioids are characteristic of a state of reduced brain arousal, i.

It is unclear, however, how the sedative properties of opioids per se may impact on breathing. In states of reduced brain arousal, such as sleep, respiratory activity depends more on the integrity of the brainstem respiratory network than it does during wakefulness 17 , Inhibition of the respiratory network by opioid ligands during states of reduced brain arousal, for instance sleep or anaesthesia, have serious effects on breathing stability and persistence 2 , 9.

In humans, similarly to sleep-disordered breathing where respiratory events mostly occur during non-rapid-eye-movement non-REM and REM sleep, respiratory depression by opioids is more pronounced during sedation Considering the powerful sedative properties of opioid drugs and their impacts on electrocortical activity, we aim to understand the relationship between changes in arousal and respiratory depression.

In addition, mechanistic 19 and drug discovery studies 20 rely on rodent models to identify new mechanisms and therapies. A better understanding of both the sedative and respiratory effects of opioids is therefore needed to better assess the potency of new opioid analgesics without the side-effect of respiratory depression. We hypothesized that sedation by fentanyl induces specific electrocortical changes that are consistent with reduced arousal but differ from the electrocortical changes observed with non-REM and REM sleep.

We also propose that the magnitude of respiratory rate depression depends on the electrocortical changes induced by opioid drugs, as previously suggested by the state-dependent effects of opioid inhibition of the medulla on breathing 9. To test these hypotheses, we determined the effects of fentanyl versus control on electrocortical and respiratory activities in freely-behaving rats. We used signal processing methods to identify common behaviours in frequencies between cortical and respiratory activities in response to the opioid analgesic fentanyl.

Spectral analyses were applied to the electrocortical signals to identify the frequency regions affected by opioid drugs. We then related these changes to respiratory rate depression to identify the cortical signatures associated with depression by opioids.

To detect common behaviors in frequencies between electrocortical and respiratory activities, we used wavelet cross spectrum and associated coherence functions In a separate set of experiments, we induced anaesthesia in rats and determined whether levels of anaesthesia modified electrocortical activity and how these changes can affect the magnitude of respiratory depression by opioid analgesics.

All procedures were performed in accordance with the recommendations of the Canadian Council on Animal Care and were approved by the University of Toronto Animal Care Committee. Eight rats were used for control saline injection and eight rats for fentanyl injection. The experimental procedures were adapted from a previous study 9.

To record diaphragm activity, two wires were sutured onto the costal diaphragm via an abdominal approach. The rat was placed in the prone position in a stereotaxic apparatus Model SAS with blunt ear bars, and three holes were drilled into the skull for the placement of the electrocortical electrodes. Holes were drilled without damaging the dura. Insulated multi-stranded stainless steel wires were also sutured on the dorsal neck muscles to record the electromyogram. The rats recovered for one week prior to experiment.

On the day of the experiment, the rat was connected to the recording apparatus through a tether cable which allowed electrophysiological signals to be recorded while the rat moved freely in a plexiglass bowl filled with fresh bedding.

The plexiglass bowl was placed on a rotating turntable Raturn, BASi, West Lafayette, IN, United States which automatically adjusts its position when the rat moves to avoid entanglements of recording cable. During that time, quality of the signals were checked. A two-hour acclimatization period is usually long enough in rats as they are not overtly stressed or anxious.

Rats usually sleep quite well in the chamber after a few hours. Respiratory rate, diaphragm and neck muscle amplitudes were averaged every sec time bin. Averaged baseline values were then recorded over a minute period before fentanyl was injected. The dose used was however well under neurotoxic levels in rats We used systemic intraperitoneal injection to avoid severe respiratory depression and respiratory arrest that my lead to brain hypoxemia.

In another group of rats, saline control was injected under the same conditions to minimize the confounding effects on behaviours due to drug injection and animal handling. After initially increasing neck muscle activity, presumably as a result of the acute behavioural response to handling and the injection itself, control injection did not further alter sleep architecture and behaviour.

B Systemic fentanyl quickly reduced motor activity and induced a persistent sedative state. C Fentanyl significantly decreased time spent awake, and increased time spent in a state of sedation, compared to time spent in non-REM sleep in the control condition. The mean duration of episodes of sleep or sedation was significantly increased by fentanyl. Also, arousals occurred significantly less with fentanyl compared to control, and periods of fentanyl-induced sedation were less fragmented by arousal than in control.

Dia, diaphragm. EMG, electromyogram. Using Matlab scripts Mathworks , the powers of the electrocortical signal were calculated using Fast Fourier Transform and power spectral density as previously described Briefly, we calculated the power spectral density estimate using the periodogram function of Matlab for each sec epoch.

We used a 4-second Hamming window which reduced the frequency resolution of our analysis to 0. These frequency bands are consistent with EEG frequency bands used in polysomnography to quantify sleep-wake states and arousal To detect common behaviors in frequencies between electrocortical and respiratory activities, we determined the wavelet cross spectrum and associated coherence function using Daubechies wavelet transform The application of the cross-spectrum wavelet transform to electrocortical and respiratory activities can reveal localized similarities in time and scale.

Because sedation significantly differs from non-REM and REM sleep 14 , we defined epochs with lack of motor activity following systemic injection of fentanyl as epochs of sedation. In anesthetized rats, we injected fentanyl intra-peritoneally while recording the electrocortical and diaphragm muscle activities. The experimental procedures were as described previously Diaphragm muscle activity was recorded using stainless steel bipolar electrodes positioned and sutured onto the right crural diaphragm.

Rats were kept warm at For the freely-behaving experiments, two-way mixed ANOVAs with sleep-wake states being a repeated factor and treatment being the between-subjects factor followed by Holm-Sidak post-hoc tests were used to determine the state-dependent effect of fentanyl versus control on each physiological variable.

One of the key-features associated with opioid analgesics is sedation. This comparison is necessary to control for injection and manipulation of the animal. The percentages of time spent in wakefulness, non-REM, and REM sleep following control injection were compared to the percentages of time spent during wakefulness and sedation after fentanyl injection.

We defined sedation as episodes of rest after fentanyl injection, i. After control injection, animals spent In contrast, after fentanyl injection, animals spent Overall, these data show that fentanyl induced a persistent sedative state with significant changes in behaviours such as less time in wakefulness, long episodes of sedation, less arousals, and less fragmented episodes of sedation compared to non-REM sleep following control injection.

Behavioural profiling identified above indicated that sedation is distinct from non-REM sleep as the animal spent more time in sedative states than it usually did in sleep, and that sedation is less fragmented than non-REM sleep.

To provide a physiologically relevant assessment of sedation, we quantified the electrocortical changes induced by fentanyl by measuring the electrocortical signal. In rodents, anaesthesia is characterized by reduced locomotor activity and distinct changes in electrocortical activity We therefore compared electrocortical spectral activities associated with fentanyl sedation with those observed in wakefulness or non-REM sleep Fig.

We performed spectral analyses of the electrocortical signal Fig. Fentanyl induces distinct electrocortical changes associated with sedation. A Electrocortical power spectral density and power bands were calculated for each epoch. Spectrograms of electrocortical activity, i. There was no significant difference in the amplitude of neck muscle activity between states.

The average respiratory variables measured over min periods, however, can potentially miss the transient events occurring over shorter time periods.

We first observed that for some 1-min periods, respiratory rate was unchanged, whereas for others respiratory rate was significantly reduced by fentanyl Fig. We then looked at the correlations between electrocortical band power changes and respiratory rate changes. We identified significant correlations Fig. Associations between electrocortical spectral changes and respiratory rate depression induced by fentanyl.

B Representative epochs showing electrocortical, spectrogram and diaphragm muscle activities showed some epochs did not present respiratory rate depression whereas others did. Studies were conducted on all mice generated; six cohorts of animals.

After respiration was measured, mice were sacrificed and injection sites were validated before inclusion of the data for further statistical analysis. We first conducted a Shapiro-Wilk normality on the average values averaged across breaths of the pre- and post-morphine respiratory parameters e.

In comparisons of Oprm1 -deleted vs. Sham conditions a mixed-repeated measure two-way ANOVA was performed to determine if these two groups were significantly different. Oprm1 deleted or intact vs. Sham conditions. Immediately after the flick, the tail was removed from the bath.

If the tail did not flick within 10 s, then the tail was removed. The procedure was video recorded so time to response could be quantified post-hoc. Each mouse was recorded for two saline and two morphine trials. Bilateral stereotaxic injections were performed in mice anesthetized by isoflurane. After injection of the virus, mice recovered for at least 3—4 weeks before breathing metrics were recorded again.

In a subset of animals, mice were then subject to a second site deletion of the complementary brain area, ie. These mice were then allowed to recover for another period of at least 3—4 weeks, after which a third set of breathing metrics were recorded. The rostral portion of the slice was taken once the compact nucleus ambiguus was visualized.

The dorsal side of each slice containing the closing of the 4 th ventricle. Slices equilibrated for 20 min before experiments were started.

After equilibration, 20 min. Activity was recorded for 20 min. Rhythmic activity was normalized to the first control recording for dose response curves. The final libraries were sequenced on HiSeq For analysis, sequencing reads were processed using the 10x Genomics Cell Ranger v. A total of cells were sequenced. Further analysis was performed using Seurat v2. Cells with less than genes were removed from the dataset.

Data was LogNormalized and scaled at 1e4. Highly variable genes were identified and used for principal component analysis. FindAllMarkers and violin plots of known cell type markers were used to identify each cluster. Summary data generated in this study are included as a supplemental supporting file. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

This is a technically sophisticated study employing mouse transgenic approaches, viral vector-based neuronal targeting, in vivo behavioral analyses, single cell transcriptomics, and in vitro electrophysiological measurements to explore the mechanisms underlying opioid respiratory depression.

The tour-de-force of impressive tools and the clear presentation make this paper especially appealing to a wide audience beyond those who study respiration.

Thank you for submitting your article "Opioids depress breathing through two small brainstem sites" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Jan-Marino Ramirez as the Reviewing Editor and Reviewer 1, and the evaluation has been overseen by Eve Marder as the Senior Editor.

The following individuals involved in review of your submission have agreed to reveal their identity: Gaspard Montandon Reviewer 2 ; Jeffrey C. Smith Reviewer 3. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The experimental approaches employed are novel, providing a number of important results that more clearly define the critical brainstem sites and molecular phenotypes of neurons involved in OIRD. The paper is well written and the figures and supplementary material very nicely illustrate the essential experimental results.

However, there are several concerns that need to be addressed. Yes, it would be nice if there were a simple gold standard. However, this is not the reality. Gold standard for OIRD should come from a consensus of studies showing similar results. Also the "gold standard" proposed here is based upon flow which cannot be reliably measured with whole-body plethysmography.

So don't state that you are introducing a gold standard. OIRD is very state dependent and shows huge interindividual variability. Trying to imply that the respiratory rhythm is simply depressed is an oversimplification. Just look at the huge variability in Te Figure 1H. Plethysmograph recordings are extremely difficult to evaluate, because animals behave, morphine will be sedating before measuring the OIRD, this will not be a static response.

We don't understand the definition of inspiratory time, expiratory time, and pause. It seems in Figure 1G that flow doesn't cross the zero at the end of expiration as shown in the figure, but rather at the end of the pause. Is the "pause" only a longer expiration? How was this pause detected or defined? If those pauses are part of expiration, then expiratory time is also increase by morphine.

How was the criterion for pause of 0. Knowing that flow is highly variable from one animal to the other it is very likely an unreliable threshold. Along the same lines: The authors respiratory assessments in freely-behaving rodents limited for a few reasons: a using an arbitrary threshold based on flow to define the end of expiration is not reliable. Flow can change from one animal to another, so the duration of their "pause" may differ depending upon the initial baseline of the animal.

Unless the right formula is used, it is very difficult to evaluate tidal volume in rodents. In addition, mL cannot be a correct measurement in rodents without normalizing the value according to body weight.

You need to carefully consider these caveats. It is well know that sedation by opioids may affect the severity of respiratory depression so arousal levels before morphine may be an issue. Please discuss this important caveat. Under hypercapnia, arousal levels may be severely disrupted and it has been shown that arousal levels are tightly linked to respiratory depression. Hypercapnia will also affect the opiate sensitivity, and this effect will not be uniform in different areas. Carefully discuss this important caveat, and don't imply that this is an advantage, other than the fact that it regularizes the rhythm.

Figure 3—figure supplement 1. It is possible that the inflammation induced by AAV, and the damage done by the microinjection may change the response to opioids. We recommend that this should be verified with proper controls. It would be important to emphasize in the Discussion that tests need to be performed in the awake behaving mice in vivo with the transgenic lines utilized for the in vitro studies to determine if so few neurons are also responsible for OIRD in the adult system in vivo.

Thank you for resubmitting your article "Opioids depress breathing through two small brainstem sites" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Ronald Calabrese as the Senior Editor and Reviewing Editor.

The following individuals involved in review of your submission have agreed to reveal their identity: Gaspard Montandon Reviewer 2 ; Jeffrey C Smith Reviewer 3. This paper has been previously reviewed and requires some further revisions.

All these revisions can be accomplished without any further experiments. Some new analysis in needed but the vast majority of the changes involve rewriting. Estimates are only made when signal energy is below a threshold, where body motion does not obscure the thorax pressure changes. To assess the limitations of extracting breath rates this way, the algorithm was applied to 19 simultaneous recordings about 19 total hours of data on mice with the plethysmography and piezoelectric sensors.

For just the low activity intervals a little less than half the data there was a slight over estimation of 0. For the application of the algorithm to this study, estimates were performed in the low to moderate activity regions and these values were averaged over much larger intervals to provide an average respiration rate every 12 min.

This was used to obtain a baseline rate averaged over a full 24 h pretreatment and a stable estimate of percent respiratory depression post-treatment every 12 min. This interval was large enough so that even in high activity regions there were typically 30 or more intervals where estimates could be made and averaged together.

The resulting derived measures enabled a quantitative evaluation of respiratory depression, survival time and recovery time Fig. PiezoSleep output for a mouse that recovers and a mouse that fails to recover after opioid treatment. Baseline respiratory rate is first established for 24 h. Time 0, is the time at which morphine is administered. Respiratory depression is defined as the lowest percentage of baseline reached after morphine treatment purple dotted arrow. For a mouse that recovers, the green vertical arrow indicates recovery time when the respiratory rate returns to baseline.

For a mouse that does not recover, the red vertical arrow indicates survival time when breathing stops i. A bracketing approach was used to construct a dose—response curve with a minimal number of mice.

These traits are largely uncorrelated. Recovery time and survival time are mutually exclusive traits and each mouse can either recover or fail to recover, not both. These values indicate that the traits are heritable and also amenable to genetic mapping. Strain- and sex-specific effect of morphine on respiratory sensitivity.

The traits of recovery time or survival time are censored such that a mouse does not appear in both graphs as each mouse displays only one of these two phenotypes. Empty bars indicate that no mice fell into this category i. Scatterplot of percent respiratory depression vs recovery time top right panel showing a weak correlation of R 2 of 0.

Published data reveal a variety of morphine LD 50 values for mice. Yoburn et al. Using this approach, survival curves by strain and sex were established Fig. The morphine LD 50 ranged from There was not a consistent sex bias up or down across all strains; instead, some LD 50 values were higher in females than males e. The morphine LD 50 by strain and sex.

The morphine LD 50 was determined for each of the eight founder strains and sex using at least six mice in each group and at least three doses of morphine, with at least two doses flanking the LD A Logistic 2-paramater survival curves separated by strain and sex are shown at the top and composites of all strains separated by sex are shown at the bottom. JMP To find genetic loci that influence OIRD, quantitative trait loci QTL mapping was performed on the respiratory phenotypes using the high diversity, high precision, DO mouse population.

Of the DO mice entered into the study, 83 females, males recovered and 67 females, 40 males did not recover. The quantitative metrics for respiratory response to morphine, including respiratory depression, recovery time and survival time, all show a continuum of phenotypic diversity Fig. Respiratory response to morphine in Diversity Outbred mice. The distribution of respiratory responses is shown for respiratory depression top panel , recovery time middle panel and survival time bottom panel.

A significant QTL was identified for respiratory depression, but no genome-wide significant QTLs were detected for recovery time or survival time using these sample sizes. For the respiratory depression trait, we identified a LOD 9. The QTL is called Rdro1 respiratory depression, response to opioids 1.

QTL mapping of respiratory depression in DO mice. B Allele effect plot of the LOD 9. Genetic mapping studies are used to identify regions of interest containing variants that influence complex traits.

To identify the relevant genes involved in complex trait regulatory mechanisms, there must be evidence of genetic polymorphisms segregating in the population that either influence protein structure or gene expression and evidence of a biological mechanism of action connecting them to the trait, such as expression in a trait-relevant tissue.

We identified a While these SNPs remain candidates for regulation of the respiratory depression phenotype, we focused on coding SNPs because their impact is more readily predictable. None of the coding SNPs are the type with the most deleterious effects, such as a stop loss, stop gain or coding region insertion frameshift. Two of the three changes serine to threonine at amino acid in Kmt2c and asparagine to aspartic acid at amino acid 29 in Speer4a occurred in residues that are not conserved across species.

This amino acid is located within a functional domain that is conserved in vertebrates Fig. Indeed, this change places the hydrophobic residue, which are generally buried internally, onto the surface of the protein.

The 3D protein structure analysis Fig. A Multi-species alignment of the Ricin B lectin domain of GALNT11 showing that the SL mutation occurs in a domain conserved across multiple vertebrates, including humans, mice, rats, Xenopus , and zebrafish. The 3D structure rendered showing secondary structure as a cartoon type with coloring as a rainbow from N- to C-terminus. In this study, we found heritable strain differences in the quantitative metrics of respiratory response to morphine, including respiratory depression, recovery time and survival time, using an advanced, high-throughput, behavioral phenotyping protocol.

We further identified genomic loci involved in morphine-induced respiratory depression using an unbiased genetic approach. Mapping these traits in the DO mice and evaluation of sequence variants and protein structure, followed by integrative functional genomic analysis in GeneWeaver, has allowed us to implicate Galnt11 as a candidate gene for respiratory depression in response to morphine.

We identified specific inbred strains of mice that were more sensitive to morphine than other inbred strains of mice. The traits of respiratory depression, recovery time and survival time were all shown to have a high degree of heritability.

In determining our probe dose for the outbred population, we observed that the LD 50 for morphine differed by four-fold between these eight parental strains harboring 45 million SNPs, or an equivalent genetic variation as found in the human population. The traits of respiratory depression, recovery time and survival time were largely independent traits, as seen by their lack of correlation. Strains such as AJ, which had the lowest LD 50 for both males and females, did not demonstrate the highest degree of respiratory depression, suggesting that factors other than respiratory depression may play a role in opioid overdose.

The lack of correlation between percent respiratory depression and survival time suggest other mechanisms of death in conjunction with OIRD. Dolinak suggests that other baseline characteristics, such as obstructive sleep apnea, obesity, heart disease and lung disease, make an individual more susceptible to opioid toxicity The presence of the alleles segregating in the DO population are encouraging for finding additional QTLs related to recovery time and survival time in larger cohorts and possibly using alternatative opioids.

The mechanisms underlying sensitivity to morphine and fentanyl are known to differ in many respects and this same work should be performed for the more potent fentanyl. Fentanyl has similarities to morphine with respect to the recruitment of intracellular signaling mechanisms but there are also key differences. Only one study has looked at human opioid overdose risk, specifically by scoring overdose status and determining the number of times that medical treatment was needed in European American populations Human genes have thus been mapped to opioid use, opioid dependence and opioid overdose susceptibility but human studies are not able to assess opioid-induced respiratory depression, specifically the LD 50 of an opioid.

Animal studies have allowed us the opportunity to assess the LD 50 of a drug in a variety of genetic backgrounds and then map those sources of variation. These types of controlled exposure experiments cannot be conducted in humans for which exquisite control of environment is not feasible and prior exposure history is unknown. Our genetic approach of QTL mapping in the DO mouse population has allowed us to identify a genomic region containing no genes previously known to function in opioid pharmacodynamics or pharmacokinetic processes, or implicated in OUD.

The genetically diverse structure of this population allows for the identification of narrow genomic intervals often with very few candidate genes.

This approach of using advanced mouse populations together with integrative functional genomics has been useful for the prioritization of candidate genes in a variety of different disciplines 62 , 63 , The identification of Galnt11 as functioning within the morphine respiratory response reveals a potential new target for therapeutic development.

GALNT11 is an N -acetylgalactosaminyltransferase that initiates O-linked glycosylation whereby an N -acetyl- d -galactosamine residue is transferred to a serine or threonine residue on the target protein. The lectin domain of GALNT11 is the portion that functions to recognize partially glycosylated substrates and direct the glycosylation at nearby sites.

This type of post-translational modification controls many phamacokinetic and pharmacodynamic processes as well as the regulation of delta opioid receptor OPRD1 membrane insertions as O-linked glycosylation is required for proper export of OPRD1 from the ER O-linked glycosylation is also required for opioid binding peptides, increasing their ability to cross the blood brain barrier The integrative functional analysis in GeneWeaver identified Hs6st2 36 , Fn1 37 , Lrp1 36 , and Sdc4 38 as glycosylation targets of Galnt Our findings demonstrate the initial mapping of a locus involved in OIRD in mice, for which the likely candidates do not act via the opioid receptor, thereby providing a potential new target for remedial measures.

Although it is through mouse genetic variation that we identified this gene, it should be noted that this gene or its glycosylation targets need not vary in humans to be a viable target mechanism for therapeutic discovery and development.

Characterization of the role of Galnt11 and its variants along with other viable candidates will resolve the mechanism further, and continued mapping studies in larger populations will enable detection of additional loci for various aspects of the opioid-induced respiratory response.

These findings suggest that phenotypic and genetic variation in the laboratory mouse provides a useful discovery tool for identification of previously unknown biological mechanisms of OIRD.

All mice were acquired from The Jackson Laboratory JAX and were housed in duplex polycarbonate cages and maintained in a climate-controlled room under a standard light—dark cycle lights on at 0, h.

Bedding was changed weekly and mice had free access to acidified water throughout the study. Louis, MO. A Nestlet and Shepherd Shack were provided in each cage for enrichment. Mice were housed in same sex groups of three to five mice per cage. A mouse plethysmography chamber was built consisting of a ml plexiglass chamber with ports for air supply and pressure measurement, and end openings outfitted with rubber gaskets to create an airtight seal after closing.

A piezo film sensor was sealed into the bottom of the chamber for data collection with PiezoSleep software, with piezo signal sampling set at Hz. A differential pressure transducer Biopac measures the pressure difference between the plethysmography chamber and reference chamber sampled at Hz. Signal alignment was done through a simple correlation, which typically indicates time differences of several seconds.

The program graphically displays an overlay of the piezo and plethysmography signals for easy visual confirmation. A graphical breath rate overlay allows navigation to intervals of signal disagreement to inspect the signals at these areas.

Not all strains received all doses but each strain received at least three doses such that two flanked one above, one below the LD Individual testing is necessary due to the known enhanced lethality of cage mates during morphine exposure, which has been shown to affect survival The mice had access to food and water ad libitum while in the chamber. The room was maintained on a h light:dark cycle. They remained in their chambers undisturbed until 24 h after injection. Whenever possible, complete balanced cohorts of the eight strains and both sexes were run during each of nine replicates of the experiment.

The data acquisition computer, food and water were checked daily; otherwise, the mice remained undisturbed. Breath rates were estimated from 4-s intervals in which animal activity dropped low i. The respiratory rate baseline consisted of the average respiratory rate over the first 24 h, which included both sleep and wake periods. Respiratory rate was then measured in the same way after injection of opioid. These measures were then used to determine thresholds for obtaining the recovery time respiratory rate returns to baseline, see Fig.

This dose was determined as the average LD 50 dose across the eight strains and two sexes, 16 samples. To test for difference in the respiratory depression, recovery time and survival time across the strains and sexes a linear model was fit, the full model was:. In all cases, the full model was fit and reduced by dropping non-significant interactions followed by main effects. The LD 50 was calculated using the drc 3. A logistic regression model was fit, and a goodness of fit test based upon Bates and Watts 72 performed.

In addition, a regression model assuming equal LD 50 across strains was compared by chi square to an LD 50 assumed different across strains. Opioid-induced Respiratory Depression OIRD , usually caused by opioid use or post-operative complications from anesthesia, occurs when the opioids desensitize the brain stem to rises in CO2, which can rapidly lead to full-blown respiratory failure.

Given the growing number of calls due to opioid abuse and subsequent respiratory arrest, it is critical that EMS providers understand how to diagnose and treat OIRD. The only treatment for an opioid overdose is the administration of naloxone, an opioid antagonist. Quantitative waveform capnography can serve as an important tool to help EMS providers in initial assessments and to measure the administration of naloxone therapy.

If administered too quickly, naloxone can cause some patients to become violent, especially if other drugs are present in the system.

Accurate diagnosis of the cause of respiratory distress is critical in order to administer appropriate treatment, but it can be difficult when the patient is unconscious or combative.