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Xylazine
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Neuropsychopharmacology
Xylazine effects on opioid-induced brain hypoxia
Shinbe Choi, Matthew R. Irwin, Eugene A. Kiyatkin*
Behavioral Neuroscience Branch, National Institute on Drug Abuse – Intramural Research Program,
National Institutes of Health, DHHS, Baltimore, MD 21224, USA
Abbreviated title: Xylazine and its interaction with fentanyl and heroin
Text information:
Number of figures: 5
Number of words:
Abstract: 250
Introduction: 539
Methods: 1480
Results: 731
Discussion: 1388
Total = 4138
* Correspondence should be addressed to Eugene A. Kiyatkin at the above address.
Fax: (443) 740-2155; tel.: (443) 740-2844; e-mail: ekiyatki@intra.nida.nih.gov
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Xylazine
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Abstract
Xylazine has emerged in recent years as an adulterant in an increasing number of opioid-positive
overdose deaths in the United States. Although its exact role in opioid-induced overdose deaths is
largely unknown, xylazine is known to depress vital functions and cause hypotension, bradycardia,
hypothermia, and respiratory depression. In this study, we examined the brain-specific hypothermic and
hypoxic effects of xylazine and its mixtures with fentanyl and heroin in freely moving rats. In the
temperature experiment, we found that intravenous xylazine at low, human-relevant doses (0.33, 1.0,
3.0 mg/kg) dose-dependently decreases locomotor activity and induces modest but prolonged brain and
body hypothermia. In the electrochemical experiment, we found that xylazine at the same doses dose-
dependently decreases nucleus accumbens oxygenation. In contrast to relatively weak and prolonged
decreases induced by xylazine, intravenous fentanyl (20 μg/kg) and heroin (600 μg/kg) induce stronger
biphasic brain oxygen responses, with the initial rapid and strong decrease, resulting from respiratory
depression, followed by a slower, more prolonged increase reflecting a post-hypoxic compensatory
phase, with fentanyl acting much quicker than heroin. The xylazine-fentanyl mixture eliminated the
hyperoxic phase of oxygen response and prolonged brain hypoxia, suggesting xylazine-induced
attenuation of the brain’s compensatory mechanisms to counteract brain hypoxia. The xylazine-heroin
mixture strongly potentiated the initial oxygen decrease, and the pattern lacked the hyperoxic portion
of the biphasic oxygen response, suggesting more robust and prolonged brain hypoxia. These findings
suggest that xylazine exacerbates the life-threatening effects of opioids, proposing worsened brain
hypoxia as the mechanism contributing to xylazine-positive opioid-overdose deaths.
Keywords: brain hypoxia, brain hyperoxia, hypothermia, peripheral vasodilation, cerebral
vasoconstriction
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Xylazine
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Introduction
A new player in the US opioid epidemic is xylazine, which is a non-controlled substance
traditionally used as a veterinary tranquilizer and component of general anesthesia in animals [1].
Although xylazine is not approved for human use, a pattern of recreational use in the US has emerged in
the past decade [2]. Reports from the Drug Enforcement Administration show that xylazine-positive fatal
overdoses have experienced a significant jump from 2020 to 2021, especially in the South region where
the noted increase was 1127% [3]. More recently, in 2022, xylazine was found as an adulterant in a
significant percentage of opioid-related overdose deaths, appearing in 25.8% of total overdose deaths in
Philadelphia and 19.3% of total overdose deaths in Maryland [4]. Fentanyl was present in 98% and
heroin in 23% of xylazine-involved deaths, revealing a dangerous connection between xylazine and
these opioid drugs.
Xylazine’s exact role in opioid-induced overdose deaths is largely unknown. Similar to clonidine,
xylazine is an agonist at the alpha-2 adrenergic receptors that induces sedation and muscle relaxation
[5]. At higher doses, xylazine has been shown to significantly depress vital functions, causing strong
hypotension, bradycardia, hypothermia, and respiratory depression [6]. When taken with opioids that
share many common effects with xylazine, the risk of overdose and death may increase. However, the
mechanisms underlying the effects of xylazine and its interaction with opioid drugs are still relatively
unknown.
In this study, we focused on the effects of xylazine as an adulterant drug taken in combination
with heroin and fentanyl. These two highly potent opioid drugs induce strong respiratory depression and
robust brain hypoxia at low doses [7-10]. To learn more about the basic physiological and behavioral
effects of xylazine, we used multi-site thermorecording coupled with monitoring of locomotor activity.
As temperature is an important homeostatic parameter, simultaneous recordings from the brain site,
temporal muscle and subcutaneous space provides a valuable tool for clarifying the mechanisms
underlying changes in brain temperature and assessing the metabolic and vascular effects of xylazine
[11].
Since xylazine is a CNS depressant that can inhibit respiration at high doses, we used oxygen
sensors coupled with high-speed amperometry to examine the effects of xylazine on brain oxygenation.
Although the hypoxic effects of drugs can be assessed by plethysmography [7,12,13] or pulse oximetry
[14-16], it is unclear how changes in breathing activity or hemoglobin saturation in blood translate into
changes in oxygen levels in the brain’s extracellular space—a functionally important parameter that
affects the health and survival of neural cells. In contrast to these peripheral measures, oxygen sensors
allow direct assessment of real-time fluctuations in brain oxygenation induced by natural stimuli,
occurring during motivated behavior, and after exposure of different drugs in freely moving rats under
physiologically relevant conditions [11, 17-21].
After we established the basic physiological effects of xylazine, we examined how this drug at a
moderate, human-relevant dose affects changes in brain oxygenation induced by fentanyl and heroin. In
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Xylazine
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these tests we compared changes in brain oxygen levels induced by fentanyl and heroin with their
combined administration with xylazine. Like in our previous studies, the nucleus accumbens (NAc), a
deep brain structure involved in sensorimotor integration and functioning of the motivation-
reinforcement circuits [22-24], was the recording site in both thermorecording and electrochemical
experiments.
Materials and Methods
Subjects
15 adult male Long-Evans rats (Charles River Laboratories) weighing 450±50 g at the time of
surgery were used in this study. Rats were individually housed in a climate-controlled animal colony
maintained on a 12-12 hr light-dark cycle with food and water available ad libitum. All procedures were
approved by the NIDA-IRP Animal Care and Use Committee and complied with the Guide for the Care
and Use of Laboratory Animals (NIH, Publication 865-23). Maximal care was taken to minimize the
number of experimental animals and any possible discomfort or suffering at all stages of the study.
Overview of the study
This study combines data obtained with two different technologies used in freely moving rats.
By using multi-site thermorecording combined with monitoring of conventional locomotion, we
assessed the basic behavioral, metabolic, and peripheral vascular effects of xylazine at doses within a
range of possible human consumption. To assess drug-induced fluctuations in brain oxygenation, we
used oxygen sensors coupled with high-speed amperometry. First, we examined the effects of xylazine
at different doses on brain oxygenation. Second, we examined the changes in brain oxygenation induced
by a fentanyl-xylazine mixture and compared them with the effects of fentanyl alone. To assess whether
the effects of xylazine-fentanyl mixture are generalized to other opioid drugs, we examined the pattern
of oxygen fluctuations induced by co-administration of xylazine with heroin and compared them with
the effects of heroin alone.
Surgical preparations
In both thermorecording and electrochemical experiments, we used similar surgical
preparations described in detail elsewhere [25, 26]. Under general anesthesia (ketamine 80 mg/kg +
xylazine 8 mg/kg with subsequent dosing), each rat was implanted with a jugular catheter. For
thermorecording experiments, the rat was implanted with three copper-constantan thermocouple
sensers in the NAc shell, temporal muscle, and subcutaneously along the nasal ridge with the tip ~15
mm anterior to bregma. Target coordinates of the recordings in the right NAc shell were: AP +1.2 mm,
ML ±0.8 mm, and DV +7.2-7.6 mm from the skull surface, according to coordinates of the rat brain atlas
[27]. For electrochemical experiments, each rat was implanted in the same NAc location with a Pt-Ir
oxygen sensor (Model 7002-02; Pinnacle Technology, Inc., Lawrence, KS, USA). The probes were secured
with dental cement to the three stainless steel screws threaded into the skull. In both experiments, the
jugular catheter ran subcutaneously to the head mount and was secured to the same head assembly.
Rats were allowed a minimum of 5 days of post-operative recovery and at least 3 daily habituation
sessions (~6 h each) to the recording environment; jugular catheters were flushed daily with 0.2 ml
heparinized saline to maintain patency.
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Xylazine
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Electrochemical detection of oxygen
Pinnacle oxygen sensors consist of an epoxy-sheathed disc electrode that is grounded to a fine
surface using a diamond-lapping disc. These sensors are prepared from a Pt-Ir wire 180 µm in diameter,
with a sensing area of 0.025 mm2 at the tip. The active electrode is incorporated with an integrated
Ag/AgCl reference electrode. Dissolved oxygen is reduced on the active surface of these sensors, which
is held at a stable potential of -0.6 V versus the reference electrode, producing an amperometric
current. The current from the sensor is relayed to a computer via a potentiostat (Model 3104, Pinnacle
Technology) and recorded at 1-s intervals, using PAL software utility (Version 1.5.0, Pinnacle
Technology).
Oxygen sensors were calibrated at 37°C by the manufacturer (Pinnacle Technology) according to
a standard protocol described elsewhere [20]. The sensors produced incremental current changes with
increases in oxygen concentrations within the wide range of previously reported brain oxygen
concentrations (0-40 μM). Substrate sensitivity of each sensor varied from 0.57-1.19 nA/1μM. Oxygen
sensors were also tested by the manufacturer for their selectivity toward other electroactive
substances, including dopamine (0.4 μM) and ascorbate (250 μM), none of which had significant effects
on reduction currents.
Experimental procedures
A similar protocol was utilized in both thermorecording and electrochemical experiments. At the
beginning of each experimental session, rats were minimally anesthetized (<2 min) with isoflurane and
sensors (either thermocouple or oxygen) were connected via an electrically shielded flexible cable and a
multi-channel electrical swivel to the recording instruments. The injection port of the jugular catheter
on the head mount was connected to a plastic catheter extension that allowed stress and cue-free drug
delivery from outside the cage. When the rats received two different drugs within one recording
session, two catheter extensions mounted on the recording cable were used to minimize any
contamination of one drug by another drug. Testing began a minimum of 90 min after connecting the
sensors to the recording instruments, when baseline values of temperature and electrochemical
currents stabilized. For the next 4-6 hours, rats received one of three drug treatments. Upon completion
of drug treatments, rats were removed from the cages and briefly anesthetized by isoflurane to
disconnect them from the recording instruments. Then, catheters were flushed with heparinized saline
before rats were returned to the animal colony. Temperature recordings were combined with
monitoring of locomotor activity using 4 infrared motion detectors (Med Associates) as previously
described [25].
The recordings were conducted for several sessions (n=3-6), and the number of sessions in each
experiment was determined by the quality of the recording and patency of the iv catheter over time. In
the thermorecording experiment, we examined changes in temperatures and locomotion induced by iv
xylazine (Xylazine hydrochloride, MP) at three doses (0.33, 1 and 3 mg/kg). These doses are lower than
the generally accepted range of toxic effects of oral xylazine that vary in humans, between 40 and 2400
mg (or 0.6 and 34.3 mg /70 kg). The largest dose used (3 mg/kg) was well below the LD50 for iv xylazine
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Xylazine
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in rats, which is between 22-43 mg/kg [28]. The drug was administered in an ascending order, with 60-
and 90-min inter-injection intervals for the 0.33 and 1 mg/kg doses. Xylazine was delivered in 0.15-0.8
ml volumes of saline at a slow injection rate (0.2 ml/10 s).
Three types of tests were conducted in the electrochemical experiments. First, we mimicked the
protocol of the thermorecording experiment and examined the effects of xylazine at the same three
doses on NAc oxygenation. During the second treatment protocol, the rat received two iv injections of
fentanyl (Fentanyl citrate injection 50 µg/mL; Fentanyl Citrate Injections; Hospira Inc.) at a 20 µg/kg
dose both alone and as a mixture with xylazine at an effective dose determined in the first treatment
protocol. As shown previously [9], 20 µg/kg is a modest dose that induces a biphasic brain oxygen
response, with a rapid and strong decrease (to ~50% of baseline levels in drug-naïve rats) followed by
more prolonged and weaker oxygen increase. In the third protocol, rats received two injections of
heroin (Diamorphine Hydrochloride; obtained from NIDA-IRP Pharmacy) at a 600 µg/kg dose, both alone
and as a mixture with xylazine at the same dose as in the second experiment. A 600 ug/kg dose is much
larger than the optimal dose for heroin self-administration (75-100 µg/kg; [29]), but it induces a similar
degree of decrease (~50% decrease) in NAc oxygen [30]. This dose difference is within the range of
generally accepted differences in potencies of fentanyl and heroin to maintain iv self-administration
behavior (ED50 2.5 ug/kg vs. 50 µg/kg or 1:20; [31]. In both the second and third treatment protocols,
the second injection (xylazine-fentanyl or xylazine-heroin) was done 120 min after the first injection
(fentanyl or heroin alone). The first and second drug protocols were conducted in the same rats and
third protocol was conducted in a separate group of rats.
Histological verification of electrode placements
After completion of the experiments, rats were sacrificed, and their brains were extracted and
placed in 10% formalin. Brains were sliced on a cryostat and analyzed for verification of the location of
cerebral implants as well as possible tissue damage around the recording site.
Data analysis
Temperature data were sampled at 2-s time intervals and analyzed with 1-min time beans. They
were presented as both absolute and relative changes with respect to the moment of drug
administration. We also calculated NAc-muscle and skin-muscle temperature gradients that were used
to determine the effects of xylazine on brain metabolic activity and tone of skin vasculature. Locomotor
data were assessed with 1-min time resolution. Electrochemical data were sampled at 1 Hz using PAL
software utility (Pinnacle Technology) and analyzed with 1-min and 10-s time resolutions.
Electrochemical data were first analyzed as raw currents. Because each individual sensor differed slightly
in background current and substrate sensitivity in vitro, currents were transformed into concentrations
and represented as relative changes, with the pre-stimulus baseline set at 100%. One-way repeated
measures ANOVAs (followed by Fisher LSD post-hoc tests) were used to evaluate statistical significance
of drug-induced changes in temperature and brain oxygen changes. Two-way repeated-measure
ANOVAs were used to analyze between-group differences in the effects of xylazine and its mixture with
fentanyl and heroin.
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Xylazine
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Results
1. Behavioral and temperature effects of xylazine
Xylazine, within the range of chosen doses, had sedative effects, decreasing temperature in all
recording locations (Figure 1). Consistent with our previous studies [31], basal temperature was highest
in the NAc, lower in temporal muscle, and lowest in the subcutaneous space (A). When analyzed as
relative changes (B), temperature decreases were significant (see F values in Supplementary materials)
and clearly dose-dependent. Despite parallel changes, the decrease was strongest in temporal muscle,
weaker in the NAc, and lowest in the subcutaneous space. Due to these differences, NAc-muscle and
skin-muscle temperature differentials significantly increased, with weaker changes for the former and
much stronger changes for the latter (C). Figure 1D shows that xylazine tended to decrease locomotor
activity; this effect was less evident at the lowest dose and more evident with higher drug doses.
-----------
Figure 1
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2. Effects of xylazine on NAc oxygenation
The effects of xylazine on NAc oxygenation were examined in 5 rats during 11 daily sessions. As
shown in Figure 2A, xylazine at each dose rapidly decreased NAc oxygen levels, then followed with a
slower ascent to baseline. Xylazine-induced oxygen responses were clearly dose-dependent. At the
lowest dose (0.33 mg/kg), oxygen decrease was minimal in both its magnitude and duration, but
stronger and more prolonged at higher drug doses (1.0 and 3.0 mg/kg). The rapidity of oxygen response
was evident when data were analyzed with rapid, 10-s time resolution for 10 min post-injection (B). In
this case, the largest drop in oxygen levels at each dose occurred within 10-30 s from the injection
onset, i.e., within the duration of drug delivery.
-----------
Figure 2
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3. Effects of xylazine on changes in NAc oxygenation induced by fentanyl
Next, we assessed the effects of a mixture of xylazine at a modest dose (1 mg/kg) on oxygen
responses induced by fentanyl (20 µg/kg). These tests were conducted in 5 rats during 16 daily sessions.
Consistent with our previous studies [9], fentanyl induced a biphasic NAc oxygen response (F15,1850=12.4,
p<0.001) with a rapid and strong decrease (~62% below pre-injection baseline) followed by a more
prolonged and weaker increase (Figure 3A).
The xylazine-fentanyl mixture also induced an oxygen decrease (F10,1210=12.4, p<0.001), which
mirrored the hypoxic response from administering fentanyl alone (~63% below the pre-injection
baseline), but lacked the second phase of the oxygen response (Figure 3A). As shown by using a two-way
ANOVA with repeated measures, between group-differences were significant from 4 to 22 min (Time x
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Xylazine
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Treatment Interaction F130,3120=3.49, p<0.001). Between-group differences were especially evident when
the data were analyzed with rapid, 10-s time resolution (Figure 3B). Due to disappearance of the second
phase of oxygen response, the total duration of oxygen decrease was longer with the drug mixture than
with the fentanyl alone (770 s vs. 330 s). Both fentanyl alone and its mixture with xylazine induced
similar behavioral effects, including severe hypoactivity, muscle rigidity in the limbs, tail erection as well
as decreases in rate and depth of respiration.
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Figure 3
------------
In most cases (10/13), the fentanyl-xylazine mixture induced similar, sedative behavioral effects
and the same monophasic pattern of oxygen response (see original examples of oxygen changes induced
by fentanyl alone and its mixture with xylazine in Figure 4A and B). However, in three cases (in 2 rats),
the xylazine-fentanyl mixture produced convulsions within a couple of minutes post-injection. In this
case, oxygen levels strongly decreased but robustly fluctuated in association with convulsion episodes
(Figure 4C and D). These three cases were analyzed separately from the main data set.
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Figure 4
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4. Effects of xylazine on changes in NAc oxygenation induced by heroin
The effects of xylazine on NAc oxygenation were tested in 6 rats during 14 daily sessions. As
shown in Figure 5, heroin (600 ug/kg) administered alone induced a robust and prolonged decrease in
NAc oxygen levels (75.0±3.9 % of baseline for ~17 min) followed by a weaker, more prolonged oxygen
increase. The xylazine-heroin mixture also strongly decreased brain oxygenation and this decrease was
clearly stronger (61.6±3.0 %; t=2.06, p<0.05) and more prolonged (~72 min) than with heroin alone. As
shown by used a two-way ANOVA with repeated measures, the between-group difference was
significant (Time x Treatment Interaction F130,3250=2.55, p<0.001). Between-group differences in oxygen
response were especially evident when we analyzed the areas under the curve for oxygen decrease
(4461.1±138.1 vs. 1124±153.6, t=16.2; p<0.0001).
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Figure 5
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Discussion
We examined the effects of xylazine as an adulterant to highly potent opioids fentanyl and
heroin. Since brain hypoxia following respiratory depression is the most dangerous effect of opioid drugs
[7, 10, 32, 33], we employed oxygen sensors coupled with high-speed amperometry to examine how
xylazine at relatively low, human-relevant doses affects brain oxygenation and whether the addition of
xylazine affects the hypoxic effects of fentanyl and heroin, the two drugs largely implicated in overdose-
related health complications and death.
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Xylazine
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Alpha-2 adrenoceptors, the primary substrate for xylazine action, are expressed in both the
central and peripheral nervous systems [34-36]. By preferential presynaptic location on central neurons,
stimulation of these receptors inhibits the release of norepinephrine, decreasing sympathetic activity
and inducing CNS depression. These receptors are also located on smooth muscle cells in blood vessels,
and their stimulation induces muscular atonia and decreases vascular tone. Stimulation of these
multiple receptors appears to be responsible for a plethora of physiological effects of xylazine, which
also depend on the dose and route of administration. In contrast to the relatively large doses of xylazine
used during general anesthesia in animals (~10 mg/kg with ip administration in rats), doses used by
humans are highly variable and the drug is delivered orally, subcutaneously, and intravenously. The
range of toxic doses in humans also greatly varies from 40 to 2400 mg (or 0.6-34.3 mg/70 kg). Since
xylazine is rarely used alone and its dosage as adulterant to other more potent drugs is typically low, we
chose to test the effects of this drug at relatively low iv doses (0.33-3.0 mg/kg), much lower than the
LD50 for iv injections in rats (22-43 mg/kg) [28].
Despite this low-dose exposure, xylazine induced evident sedation, muscle relaxation, and
hypothermia—known effects of the drug [37, 38]. In contrast to older studies employing rectal
temperature measurements, we used chronically implanted thermocouple sensors, stress-free drug
delivery, and high-resolution data analyses to reveal differences in xylazine-induced temperature
changes in the brain, temporal muscle, and skin. Although basal temperature in the brain was larger
than in temporal muscle, the xylazine-induced decrease was stronger in the muscle than in the brain.
This pattern is unusual since temperature changes induced by natural arousing stimuli are typically more
rapid and stronger in the brain than in the temporal muscle, suggesting an increase in intra-brain heat
production, a sequence of metabolic neural activation [11]. While we could not fully exclude that
xylazine may increase metabolic brain activity, stronger temperature decreases in temporal muscle
likely result from muscular atonia and atonia-related decreases in heat production [39]. The xylazine-
induced temperature decrease in the subcutaneous space was weaker than in the muscle, resulting in a
strong increase in the skin-muscle differential. While this change indicates skin vasodilation as a primary
reason for heat loss and resulting brain and body hypothermia, we cannot exclude the possibility of an
atonia-related decrease in muscular heat production.
Xylazine at large doses can induce respiratory depression and life-threatening hypoxia in animals
[40, 41]. Our data revealed that low doses of xylazine delivered iv decreases oxygen levels in the brain, a
central effect that may result from respiratory depression. While the effect was weak (93%, 88%, and
82% of baseline levels for 0.33, 1.0, and 3 mg/kg doses), it was prolonged and dose-dependent. This
tonic decrease in brain oxygen levels differed from the much stronger but transient decreases induced
by fentanyl and heroin, suggesting different underlying mechanisms. These moderate decreases in brain
oxygenation may also result from drug-induced decreases in sympathetic activity due to alpha-2
agonism and subsequent tonic decreases in respiratory activity. Alternatively, the weak hypoxic effect of
xylazine may be independent of respiratory depression, resulting from cerebral vasoconstriction due to
the stimulation of alpha-2 adrenoceptors on cerebral vessels [42].
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Xylazine
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The known physiological effects of xylazine and its prevalence in opioid-related overdose deaths
led us to study the effects of this drug in combination with opioids. The potentiating effect of xylazine
and other alpha-2 agonists has been found in early studies on the analgesic effects of opioid drugs,
including fentanyl [43]. However, these studies use larger doses of xylazine, and it is still under debate
whether the weakening of nociceptive responses reflects the enhancement of opioid analgesia or
reinforced sedation by xylazine. To test for the same potentiation effect, we examined xylazine effects
at a relatively low, human-relevant dose (1.0 mg/kg) on brain oxygen responses induced by fentanyl and
heroin.
We found that the addition of xylazine dramatically changes the pattern of brain oxygen
response induced by fentanyl. In contrast to the biphasic effect of fentanyl, with a rapid and strong
oxygen decrease followed by its rebound-like increase, the fentanyl-xylazine mixture resulted in
elimination of the secondary increase, prolonging the duration of the initial decrease (Fig. 3). Stronger
potentiating effects of xylazine were found on decreases in brain oxygenation induced by heroin at a
relatively large dose (600 ug/kg; 30:1 ratio vs. fentanyl). Like fentanyl, heroin also induced biphasic
oxygen responses, but both the initial decreases and subsequent increases were much more prolonged
than with fentanyl. In contrast to heroin alone, the mixture with xylazine induced stronger and more
prolonged decreases in brain oxygenation and eliminated the second hyperoxic phase of brain oxygen
response.
To determine the possible mechanisms underlying xylazine’s ability to potentiate hypoxic effects
of opioid drugs, we need to understand the mechanisms underlying the biphasic changes in oxygen
induced by these drugs. In contrast to peripheral tissues, where heroin and fentanyl induce monophasic
and relatively prolonged oxygen decreases, the brain hosts two-part oxygen responses with a strong,
transient oxygen decrease followed by a weaker, prolonged oxygen increase [26, 44]. While the initial
decrease results from respiratory depression and subsequent drop in oxygen in the blood, the
subsequent increase may result from cerebral vasodilation and increased vertebral blood flow due to
post-hypoxic accumulation of CO2, a powerful vasoconstrictor, in the brain [45-47]. Another factor that
mediates cerebral vasodilation induced by opioid drugs is peripheral vasoconstriction that results in
redistribution of arterial blood from the periphery to the brain and heart [48]. These two factors likely
explain the cerebral vasodilation and increased cerebral blood flow that enhances oxygen entry into
brain tissue despite its drop in arterial blood. This is an adaptive mechanism to counteract severe brain
hypoxia following insufficient oxygen delivery to the brain from arterial blood.
While the exact mechanisms responsible for prolongation of fentanyl- and heroin-induced
hypoxia and disappearance of post-hypoxic hyperoxia induced by xylazine still remain hypothetical,
numerous studies point to the vascular effects of this drug in the brain, specifically the blockade of
adaptive cerebral vasodilation due to known cerebral vasoconstrictive effects of xylazine and other
alpha2 adrenergic agonists [49-51]. Xylazine-induced peripheral vasodilation, which diminishes blood
inflow to the brain, and decrease in arterial blood pressure due to xylazine-induced decreased
sympathetic outflow may also contribute to the brain-specific hypoxic effects of this drug.
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Xylazine
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One finding of this study was unexpected. While administration of the fentanyl-xylazine mixture
induced sedation in rats during most sessions, the mixture rarely induced convulsions associated with
robust fluctuations in brain oxygen levels (Fig. 4). This finding suggests that the effects of the combined
use of fentanyl and xylazine are not limited to potentiation of hypoxia and may induce other life-
threatening complications. Although the mechanisms underlying this effect of drug combination remain
unclear and require further investigation, it is known that convulsions result in robust cerebral
vasodilation/increased cerebral blood flow [52, 53], which may counteract the cerebral vasoconstriction
induced by xylazine. This explanation remains speculative and require further investigation.
Conclusions and Human Implications
Overall, our findings deepen the understanding of the involvement of xylazine adulterants in
opioid overdose-induced health complications. We highlight the damaging physiological effects of
xylazine as it pertains to substance abuse when taken in tandem with opioids by showing the effects of
drug mixtures on brain oxygenation and brain temperature. We found that xylazine addition to fentanyl
results in elimination of the brain’s compensatory mechanisms to counteract rapid brain hypoxia, and
that xylazine addition to heroin potentiates the rapid opioid-induced brain hypoxia in addition to
elimination of the following compensatory mechanisms. Our results imply that xylazine can exacerbate
the life-threatening potential of opioid use, positing worsened brain hypoxia as a potential cause of
death. It will be of clinical importance to observe whether these patterns of oxygenation following
xylazine-opioid administration hold and apply in humans.
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Xylazine
12
Acknowledgements
: The study was supported by the Intramural Research Program of the NIH, NIDA
Author contributions:
EAK: Conceptualization, Surgery procedures, Participation in experiments, Data
analyses, Writing the manuscript; SC and MRI: Performance of experiments, Data analyses, Graphic work,
Histological work, Review and editing the manuscript.
Funding:
The study was supported by the Intramural Research Program of the NIH, NIDA (NIH Grant
1ZIADA000566-12 for Dr. Eugene A. Kiyatkin).
Competing interests
: The authors have nothing to disclose.
Data availability:
Raw data and the results of their primary analyses are available on request from Dr.
Eugene A. Kiyatkin (NIDA-IRP, NIH; ekiyatki@intra.nida.nih.gov).
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Xylazine
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References
1 Reyes JC, Negron JL, Colob HM, Padilla AM, Millan, MY et al. The emerging of xylazine as a new drug of
abuse and its health consequences among drug users in Puerto Rico. J Urban Health. 2012;
89(3):519-26.
2 Kariisa, M, Patel P, Smith H, Bitting J. Xylazine detection and involvement in drug overdose deaths—
United States, 2019. MMWR Morb Mortal Wkly Rep. 2021; 70(37):1300-2.
3 U.S. Department of Justice. Drug Enforcement Administration. The growing threat of xylazine and its
mixture with illicit drugs. DEA Joint Intelligence Report. 2021.
4 Friedman J, Montero F, Bourgois P, Wahbi R, Dye D et al. Xylazine spreads across the US: A growing
component of the increasingly synthetic and polysubstance overdose crisis. Drug Alcohol
Depend. 2022; 233:109380.
5 Schwartz DD, Clark TP. 1998. Affinity of detomidine, medetomidine and xylazine for alpha-2 adrenergic
receptor subtypes. J Vet Pharmacol Ther. 1998; 21:107-11.
6 Capraro AJ, Wley JF, Tucker JR. Severe intoxication from xylazine inhalation. Pediatr Emerg Care. 2001;
17:447-8.
7 Dahan, A., Yassen, A., Bijl, H., Romberg, R., Sarton, E., Teppema, L. et al. 2005. Comparison of the
respiratory effects of intravenous buprenorphine and fentanyl in humans and rats. Br. J.
Anaesth. 94, 825-34.
8 Solis E Jr, Cameron-Burr KT, Shaham Y, Kiyatkin EA. Intravenous heroin induces rapid brain hypoxia and
hyperglycemia that precede brain metabolic response. eNeuro. 2017; 4(3) ENEURO.0151-
17.2017
9 Solis E Jr, Cameron-Burr KT, Shaham Y, Kiyatkin EA. Fentanyl-induced brain hypoxia triggers brain
hyperglycemia and biphasic changes in brain temperature. Neuropsychopharmacology. 2018;
43(4):810-9.
10 Yeadon M, Kitchen I. Opioids and respiration. Prog Neurobiol. 1989; 33(1):1-16.
11 Kiyatkin EA. Brain temperature homeostasis: physiological fluctuations and pathological shifts. Front
Biosci (Landmark Ed). 2010; 15(1):73-92.
12 Brown JH, Pleuvry BJ. Antagonism of the respiratory effects of alfentanil and fentanyl by naloxone in
the conscious rabbit. Br J Anaesth. 1981; 53(10):1033-7.
13 Seckler JM, Grossfield A, May WJ., Getsy PM, Lewis SJ. 2022. Nitrosyl factors play a vital role in the
ventilatory depressant effects of fentanyl in unanesthetized rats. Biomed Pharmacother. 2022;
146:112571.
14 Decker MJ, Conrad KP, Strohl KP. Noninvasive oximetry in the rat. Biomed Instrum Technol. 1989;
23(3):222-8.
15 Hedenqvist P, Roughan JV, Flecknell PA. Sufentanil and medetomidine anesthesia in the rat and its
reversal with atipamezole, and butorphanol. Lab Anim 2000; 34(3):244-51.
16 Baby SM, Discala JF, Gruber R, Getsy P, Cheng F et al. Tempol reverses the negative
effects of morphine on arterial blood-gasd chemistry and tissue oxygen saturation in freely-
moving rats. Front Pharmacol. 2021; 12:749084
17 Ledo A, Lourenco C, Laranjinha J, Gerhardt GA. Barbosa RM. Combined in vivo amperometric
oximetry and electrophysiology in a single sensor: a tool for epilepsy research. Anal Chem.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 3, 2023. ; https://doi.org/10.1101/2023.03.31.535103doi: bioRxiv preprint
Xylazine
14
2017;89(22):12383-90.
18 Kealy J, Bennett R, Lowry JP. Simultaneous recording of hippocampal oxygen and glucose in
real time using constant potential amperometry in the freely-moving rat. J Neurosci Methods.
2013; 215(1):110-20.
19 Lowry JP, Fillenz M. Real-time monitoring of brain energy metabolism in vivo using
microelectrochemical sensors: the effects of anesthesia. Bioelectrochemistry. 2001; 54(1):39-47.
20 Bolger FB, Bennett R, Lowry JP. An in vitro characterization comparing carbon paste and Pt
microelectrodes for real-time detection of brain tissue oxygen. Analyst. 2011; 136(19):4028-35.
21 Kiyatkin E.A., 2019. Physiological and drug-induced fluctuations in brain oxygen and glucose assessed
by substrate-sensitive sensors coupled with high-speed amperometry. In: Wilson, GS and
Michael AC, editors. Compendium of in vivo monitoring in real-time molecular neuroscience.
Volume 3: Probing brain functions, disease and Injury with enhanced optical and
electrochemical sensors. World Scientific; 2019. p. 219-50.
22 Badiani A, Belin D, Epstein D, Calu D, Shaham Y. Opiate versus psychostimulant addiction: the
differences do matter. Nat Rev Neurosci. 2011; 12(11):685-700.
23 Mogenson GJ, Jones DL, Yim CY. From motivation to action: functional interface between the limbic
system and the motor system. Prog Neurobiol. 1980; 14(2-3):69-97.
24 Wise RA, Bozarth MA. A psychomotor stimulant theory of addiction. Psychol Rev. 1987; 94(4):469-92.
25 Kiyatkin EA, Brown PL. Brain temperature change and movement activation induced by
intravenous cocaine delivered at various injection speed in rats. Psychopharmacology (Berl).
2005; 181(2): 299-308.
26 Thomas SA, Curay CM, Kiyatkin EA. 2021. Relationships between oxygen changes in the brain and
periphery following physiological activation and the action of heroin and cocaine. Sci Rep. 2021;
11(1):6355.
27 Paxinos G, Watson C. 1998. The rat brain in stereotaxic coordinates, Academic Press: San Diego.
28 Committee for veterinary medical products. Xylazine Hydrochloride: Summary report. The European
Agency for the Evaluation of Medicinal Products. 1999.
29 Gerber CJ, Wise RA. Pharmacological regulation of intravenous cocaine and heroin self-
administration in rats: A variable dose paradigm. Pharmacol Biochem Behav. 1989; 32(2):527-
31.
30 Thomas SA, Perekopskiy D, Kiyatkin EA. Cocaine added to heroin fails to affect heroin-induced
hypoxia. Brain Res. 2020; 1746:147008.
31 van Ree JM., Slangen JL, de Wied D. Intravenous self-administration of drugs in rats. J. Pharmacol Exp
Ther. 1978; 204(3):547-57.
32. Jaffe, J.H., Knapp, C.M., Ciraulo, D.A., 1997. Opiates: Clinical Aspects. In: Lowinson JH, Ruiz P,
Millman RB, Langrod JG, editors. Substance Abuse, Third edn. Baltimore: Williams & Wilkins,. p.
158-66.
33 Pattinson KT. Opioids and the control of respiration. Br J Anaesth. 2008; 100(6):747-58.
34 Drew GM. Effects of alpha-adrenergic agonists and antagonists on pre- and postsynaptically located
alpha-adrenoceptors. Eur J Pharmacol. 1976; 36(2):313-20.
35 Kobinger W. Central alpha-adrenergic systems as target for hypotensive drugs. Rev Physiol Biochem
Pharmacol. 1978; 81:39-100.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted April 3, 2023. ; https://doi.org/10.1101/2023.03.31.535103doi: bioRxiv preprint
Xylazine
15
36 Greene SA, Thrmon JC. Xylazine—a review of its pharmacology and use in veterinary medicine. J Vet
Pharmacol Ther. 1988; 11(4):295-313.
37 Hsu WH. Xylazine-induced depression and its antagonism by alpha adrenergic blocking agents. J
Pharmacol Exp Ther. 1981; 218(1):188-92.
38 Livingston A, Low J, Morris B. Effects of clonidine and xylazine on body temperature in the rat. Br J
Pharmacol. 1984; 81(1):189-93.
39 Lomo T, Eken T, Rein EB, Nja A. Body temperature control in rats by muscle tone during rest or
sleep. Acta Physiol (Oxf). 2020; 228(2):e13348.
40 Thurmon JC, Benson GJ. (1986). Anesthesia in ruminants and swine. In Anderson, DE, Rings DM,
editors. The veterinary clinics of North America: food animal practice. Philadelphia: W. B.
Saunders Company; 2009 pp. 51-71.
41 Sanford TD, Colby ED. Effect of xylazine and ketamine on blood pressure, heart rate and respiratory
rate in rabbits. Lab Anim Sci. 1980; 30(3):519-23.
42 Kanawati S, Yaksh TL, Anderson RE, Marsh. Effects of clonidine on cerebral blood flow and the
response to arterial CO2. J Cereb Blood Flow Metab. 1986; 6(3):358-65.
43 Meert TF, De Kock M. Potentiation of the analgesic properties of fentanyl-like opioids with alpha 2-
adrenoceptor agonists in rats. Anesthesiology. 1994; 81(3):677-88.
44 Curay CM, Irwin MR, Kiyatkin EA. The pattern of brain oxygen response induced by intravenous
fentanyl limits the time window of therapeutic efficacy of naloxone. Neuropharmacology, in
press
45 Battisti-Charbonney A, Fisher J, Duffin J. The cerebrovascular response to carbon dioxide in humans. J
Physiol. 2011; 589(Pt 12):3039–48.
46 Schmidt CF, Kety SS. Recent studies of cerebral blood flow and cerebral metabolism in man. Trans
Assoc Am Physicians. 1947; 60(1):52–58.
47 Kontos HA. Regulation of the cerebral circulation. Annu Rev Physiol. 1981;l 43:397–407.
48 Kiyatkin EA. 2021. Functional role of peripheral vasoconstriction: not only thermoregulation but
much more. J Integr Neurosci. 2021; 20(3):755-64.
49 Busija DW, Leffler CW. Postjunctional alpha 2-adrenoceptors in pial arteries of anesthetized newborn
pigs. Dev Pharmacol Ther. 1987; 10(1):36-46.
50 Skärby T. Pharmacological properties of prejunctional alpha-adrenoceptors in isolated feline middle
cerebral arteries; comparison with the postjunctional alpha-adrenoceptors. Acta Physiol Scand.
1984; 122(2):165-74.
51 Lee HW, Caldwell JE, Dodson B, Talke P, Howley J. The effect of clonidine on cerebral blood flow
velocity, carbon dioxide cerebral vasoreactivity, and response to increased arterial pressure in
human volunteers. Anesthesiology 1997; 87(3):553–8.
52 Penfield W, von Sántha K, Cipriani A. Cerebral blood flow during induced epileptiform seizures in
animals and man. J Neurophysiol. 1939; 2:257-67.
53 Ferlini L, Su F, Creteur J, Taccone FS, Gaspard N. Cerebral and systemic hemodynamic effect of
recurring seizures. Sci Rep. 2021; 11(1), 22209.
.CC-BY-NC-ND 4.0 International licenseavailable under a
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Xylazine
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Figure legends
Figure 1. Changes in temperature induced by iv xylazine at different doses (0.33, 1.0 and 3.0 mg/kg in
awake, freely moving rats. A = Absolute temperature changes. B = relative temperature changes; C =
Brain-muscle and skin-muscle differentials. D = Locomotor activity. Filled symbols show values
significantly different from pre-injection baseline.
Figure 2. Relative changes in NAc oxygen levels induced by xylazine at different doses (0.3, 1.0 and 3.0)
in freely moving rats. A = mean (±SEM) changes assessed with slow (1-min) time resolution. B = mean
(±SEM) changes assessed with rapid (10-s) time resolution. Filled symbols show values significantly
different from pre-injection baseline. n = numbers of averaged responses
Figure 3. Mean (±SEM) changes in NAc oxygen levels induced by fentanyl (20 ug/kg) and its mixture with
xylazine (20 ug/kg + 1 mg/kg) in freely moving rats. A = mean (±SEM) changes assessed with slow (1-
min) time resolution. B = mean (±SEM) changes assessed with rapid (10-s) time resolution. Filled
symbols show values significantly different from pre-injection baseline. n = numbers of averaged
responses. Bold horizontal lines with asterisk show time intervals, during which between-group values
were significant.
Figure 4. Primary data examples of changes in electrochemical currents (nA) induced by fentanyl and
fentanyl-xylazine mixture in freely moving rats. A = fentanyl alone (20 ug/kg), B = fentanyl (20
ug/kg)+xylazine (1 mg/kg), typical example; C and D = unusual changes induced by fentanyl-xylazine
mixture with convulsions. Values of reduction current are shown with original (1-s) time resolution, and
they were inverted. Since basal reduction currents widely varied between sensors, data were analyzed
as the change relative to basal value=100%. Convulsions were never seen after fentanyl alone, but they
occurred in 3 cases (in 2 rats) after injections of fentanyl-xylazine mixture.
Figure 5. Mean (±SEM) changes in NAc oxygen levels induced by heroin (600 ug/kg) and its mixture with
xylazine (600 ug/kg + 1 mg/kg) in freely moving rats. A = mean (±SEM) changes assessed with slow (1-
min) time resolution. B = mean (±SEM) changes assessed with rapid (10-s) time resolution. Filled
symbols show values significantly different from pre-injection baseline. n = numbers of averaged
responses. Bold horizontal lines with asterisk show time intervals, during which between-group values
were significant.
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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Xylazine
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Figures and Figure Legends
Figure 1. Changes in temperature induced by iv xylazine at different doses (0.33, 1.0 and 3.0 mg/kg in
awake, freely moving rats. A = Absolute temperature changes. B = relative temperature changes; C =
Brain-muscle and skin-muscle differentials. D = Locomotor activity. Filled symbols show values
significantly different from pre-injection baseline.
-30 0 30 60 90 120 150 180 210 240 270
34
35
36
37
38
Time (m in)
Te mp e r at u re ( °C)
NAc
Skin
Muscle
Xylazine 0.3 mg/kg Xylazine 1 mg/kg Xylazine 3 mg/kg
n=12
-2.0
-1.5
-1.0
-0.5
0.0
0.5
Tem p er a t ur e Ch a n ge ( °C)
NAc
Skin
Muscle
Xylazine 0.3 mg/kg
B
Xylazine 1 mg/kg
Xylazine 3 mg/kg
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
Tem p er a t ur e Di f fe r e nt i a l ( °C)
Brain-Muscle
Skin-Muscle
C
015 30 45 60
0
5
10
15
20
25
Time (min)
Locomotion, counts/min
D
015 30 45 60 75 90
Time (min)
015 30 45 60 75 90 105 120
Time (min)
A
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Xylazine
18
Figure 2. Relative changes in NAc oxygen levels induced by xylazine at different doses (0.3, 1.0 and 3.0)
in freely moving rats. A = mean (±SEM) changes assessed with slow (1-min) time resolution. B = mean
(±SEM) changes assessed with rapid (10-s) time resolution. Filled symbols show values significantly
different from pre-injection baseline. n = numbers of averaged responses
020 40 60
80
90
100
110
Time (min)
Change in [Oxygen] (%)
0.33 mg/kg Xylazine
n=8
020 40 60 80
Time (min)
1.0 mg/kg Xylazine
n=11
020 40 60 80 100 120
Time (min)
3.0 mg/kg Xylazine
n=7
0120 240 360 480 600
85
90
95
100
105
Time (s)
Change in [Oxygen] (%)
0120 240 360 480 600
Time (s)
0120 240 360 480 600
Time (s)
A
B
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Xylazine
19
Figure 3. Mean (±SEM) changes in NAc oxygen levels induced by fentanyl (20 ug/kg) and its mixture with
xylazine (20 ug/kg + 1 mg/kg) in freely moving rats. A = mean (±SEM) changes assessed with slow (1-
min) time resolution. B = mean (±SEM) changes assessed with rapid (10-s) time resolution. Filled
symbols show values significantly different from pre-injection baseline. n = numbers of averaged
responses. Bold horizontal lines with asterisk show time intervals, during which between-group values
were significant.
020 40 60 80 100 120
60
80
100
120
140
Time (min)
Change in [Oxygen] (%)
Fentanyl + Xylazine (n=11)
Fentanyl (n=16)
✱
0300 600 900 1200 1500 1800
Time (s)
Fentanyl (n=16)
Fentanyl + Xylazine (n=11)
✱
A B
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Xylazine
20
Figure 4. Primary data examples of changes in electrochemical currents (nA) induced by fentanyl and
fentanyl-xylazine mixture in freely moving rats. A = fentanyl alone (20 ug/kg), B = fentanyl (20
ug/kg)+xylazine (1 mg/kg), typical example; C and D = unusual changes induced by fentanyl-xylazine
mixture with convulsions. Values of reduction current are shown with original (1-s) time resolution, and
they were inverted. Since basal reduction currents widely varied between sensors, data were analyzed
as the change relative to basal value=100%. Convulsions were never seen after fentanyl alone, but they
occurred in 3 cases (in 2 rats) after injections of fentanyl-xylazine mixture.
-180 0 180 360 540 720 900 1080
10
15
20
25
30
Time (s)
Current (nA)
Fentanyl + Xylazine
-180 0 180 360 540 720 900 1080
10
15
20
25
Time (s)
Current (nA)
Fentanyl + Xylazine: Convulsions 2
-180 0 180 360 540 720 900 1080
0
5
10
Time (s)
Current (nA)
Fentanyl + Xylazine: Convulsions 1
-180 0 180 360 540 720 900 1080
15
25
35
45
Time (s)
Current (nA)
Fentanyl
AB
C D
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Xylazine
21
Figure 5. Mean (±SEM) changes in NAc oxygen levels induced by heroin (600 ug/kg) and its mixture with
xylazine (600 ug/kg + 1 mg/kg) in freely moving rats. A = mean (±SEM) changes assessed with slow (1-
min) time resolution. B = mean (±SEM) changes assessed with rapid (10-s) time resolution. Filled
symbols show values significantly different from pre-injection baseline. n = numbers of averaged
responses. Bold horizontal lines with asterisk show time intervals, during which between-group values
were significant.
020 40 60 80 100 120
60
80
100
120
Time (min)
Change in [Oxygen] (%)
Heroin (n=13)
Heroin + Xylazine (n=14)
✱
0360 720 1080 1440 1800
Time (s)
Heroin (n=13)
Heroin + Xylazine (n=14)
AB
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