Human use of these devices for ingestion of cannabis extracts or cannabidiol is becoming so popular that terming these devices e-cigarettes or ENDS is becoming a misnomer; Electronic Drug Delivery Systems is more accurate. The primary goal of this study was to establish methods for delivering physiologically and behaviorally significant amounts of heroin to rats via vapor inhalation. To this end we adapted an e-cigarette based vaporization system we have previously shown is effective for the delivery of nicotine , THC , cannabidiol , methamphetamine , 3,4-methylenedioxypyrovalerone and oxycodone to rats. Other work has demonstrated antinociceptive effects of inhaled oxycodone in male rats and a preliminary work demonstrates antinociceptive efficacy of inhaled heroin and methadone in male and female rats, and reinforcing effects of inhaled heroin in female rats. Nearly identical systems have been shown to deliver nicotine , THC from cannabis extracts and the potent synthetic opioids sufentanil and fentanyl , to laboratory rodents. To validate the delivery of heroin by vapor inhalation, we used a warm water tail-withdrawal assay for assessing effects on nociception, and a radiotelemetry system capable of reporting body temperature and activity responses. The telemetry measures were selected based on prior evidence that parenteral injection of opioids causes hyperthermia and increased locomotor activity in both rats and mice. For example, intravenous morphine increased the rectal temperature of anesthetized rats , as did intrathecal morphine in freely moving male Wistar rats measured with telemetry . Both morphine and oxycodone produce hyperthermia when injected subcutaneously in rats . Solis and colleagues showed that intravenous fentanyl initially decreased body temperature of rats in the first hour after administration, did not, but each drug induced hyperthermia in the interval approximately 60–120 minutes after injection .
Heroin has also been shown to increase the locomotor activity of rats when injected subcutaneously or intraperitoneally in doses of ~0.25–1.0 mg/kg, although in some early studies a contrast of this described effect with vehicle injection was not made clear . Nevertheless,vertical growing racks system in other studies it was shown that heroin injection ~0.25–0.5 mg/kg, s.c., significantly increases the locomotor activity of rats in comparison with a vehicle injection. A secondary goal of this study was to determine the parameters necessary for dose control / manipulation within an effective range. As we’ve shown in prior studies , this can be effected with changes in drug concentration in the vapor vehicle and the duration of exposure, thus these parameters were under investigation. The final goal was to determine if there are any substantive sex differences in the response to heroin inhalation, see Craft for review, consistent with current suggestions for the inclusion of both male and female subjects in research where possible . While male and female rats acquire intravenous oxycodone self-administration at similar rates with 1-h access sessions , our recent study with 8-h access found that female rats self-administered more oxycodone in acquisition, but did not reach higher breakpoints in a PR dose-substitution . A prior study reported no sex difference in the anti-nociceptive effect of heroin or oxycodone injected subcutaneously in Sprague-Dawley rats , thus no sex difference was predicted for this study. The rats were implanted with sterile radiotelemetry transmitters in the abdominal cavity as previously described on Post-Natal Day 31. For studies, animals were evaluated in clean standard plastic homecages in a dark testing room, separate from the vivarium, during the dark cycle. Radiotelemetry transmissions were collected via telemetry receiver plates placed under the cages as described in prior investigations . Test sessions for exposure studies started with a 15-minute interval to ensure data collection, then a 15-minute interval for baseline temperature and activity values. The rats were then moved to a separate room for the vapor exposure sessions and then returned to the recording chambers for up to 300 minutes after the start of vapor exposure.
These experiments were conducted over the adult age interval of PND. The subjects had participated in similar telemetry recording experiments following vapor and injected exposure to doses of nicotine and THC from PND 50 to PND 190 with active dosing conducted no more frequently than two times per week, in studies not previously reported. The telemeterized body temperature and activity rate were collected on a 5-minute schedule in telemetry studies but are expressed as the average of six sequential measurements for primary analysis. The time courses for data collection are expressed relative to the initiation of vapor exposure and times in the figures refer to the end of the interval . Any missing temperature values were interpolated from the values before and after the lost time point. Activity rate values were not interpolated because 5-minute to 5-minute values can change dramatically, thus there is no justification for interpolating. Data for the first time-point after the start was inadvertently missing for two female rats in the 30-minute PG condition. The values for the second time point were used in place of the missing values. These ended up being very similar to the respective values recorded for this time point for the 15-minute PG condition. The telemetry data were generally analyzed with two-way Analysis of Variance including repeated measures factors for the Vapor condition and the Time after vapor initiation. A mixed-effects model was used instead of ANOVA wherever there were missing data points. The nociception data were analyzed by three-way ANOVA including repeated-measures factors for Duration of exposure and Vapor condition and a between-groups factor of Sex. Any significant main effects were followed with posthoc analysis using Tukey or Sidak procedures. All analysis used Prism 8 for Windows . This study confirms that this method of inhalation of heroin, based on vapor created by an Electronic Drug Delivery System device, produces significant effects in vivo in both male and female rats. The effects are dose-dependent, as they vary with both inhalation duration and with drug concentration in the e-cigarette vehicle .
We have previously shown that heroin vapor exposure produces moderate anti-nociceptive effects in male and female Wistar rats and that is extended to Sprague-Dawley rats, and to a wider range of inhalation conditions, in the present study. As we’ve previously shown, differences between common laboratory rat strains can result in quantitative or even qualitative differences in response to vapor inhalation of drugs, effects which may vary depending on the outcome measure . The present results obtained near-maximal antinociceptive effects in more animals in the 100 mg/mL condition, compared with that prior study, however there were equipment differences between the two studies which may have resulted in more effective drug delivery. For example that prior study reported no antinociceptive effect of oxycodone vapor inhalation using first generation Protank 3 canisters, whereas a study using the second-generation Herakles Sub Ohm canisters, more similar to the present second-generation SMOK canisters,vertical marijuana grow did report anti-nociception subsequent to 100 mg/mL oxycodone vapor inhalation . Although vapor cloud density and chamber fill can be roughly equated by eye, it may be the case that different devices generate different droplet sizes or drug concentrations per droplet that produce complex interactions of a given drug with a given device to effect in vivo drug delivery. In that prior study a 1 mg/kg, s.c., heroin injection produced antinociception comparable to the effects of 30 minutes of 50–100 mg/mL exposure in this study. The data also show that the threshold for statistically reliable effects is 15-minutes of exposure to the 50 mg/mL condition. These exposure parameters led to significant antinociception and produced significant effects on body temperature in the female rats. This study therefore establishes 50–100 mg/mL as an effective concentration range, and 1 mg/mL as an ineffective concentration, for 15–30 minutes of non-contingent inhalation exposure to heroin in rats. This is a nontrivial advance from the prior demonstrations that rats and mice, respectively, will self-administer the potent opioids sufentanil and fentanyl . First, the doses needed to produce robust changes in nociception, thermoregulation and locomotor behavior are often far in excess of the doses that rodents will self-administer. Second, we found previously that cocaine and some amphetamine or cathinone class psychomotor stimulants which exhibit both low potency and lower solubility in PG may not readily be delivered in active doses with this approach . Thus, it was critical to show that a less potent opioid such as heroin can be delivered with this method. There were biphasic dose-dependent effects of inhaled heroin on body temperature which was expressed as lower exposure conditions producing reliable increases in temperature, and higher doses / exposures initially reducing body temperature, at least in the female rats. A prior study found similar biphasic effects after intravenous fentanyl, which initially decreased body temperature of male Long-Evans rats in the first hour after administration, but induced hyperthermia thereafter . This may reflect potency, or brain penetration speed, as potential differences between the two opioids that permitted this initial hypothermia to be observed.
It could similarly be the case in the present study that the inhaled route of administration speeds the brain entry of heroin versus the more common s.c. or i.p. or even i.v. routes of administration. The observation that injected heroin did not have immediate effects on female rats’ body temperature , and induced less complete suppression of activity compared with inhaled heroin , supports this interpretation. If so, this may be a key area in which the development of this inhalation model allows the investigation of effects of heroin which are unique to this route of administration. It may also be the case that effects of inhalation are more similar to the effects of the intravenous route, in this case the method offers several practical advantages over implanting and maintaining intravenous catheters in rodents. Such advantages include overall cost, surgical expertise and avoiding subject loss due to occluded catheters or health complications related to the catheter. The apparent sex difference is most likely a difference in effective dose when heroin is inhaled, since pilot work with a group of male Sprague-Dawley rats illustrated a consistent hypothermia associated with a high exposure condition and hyperthermia with a lower exposure condition . Also, female rats in this study were affected in terms of body temperature and anti-nociception after 15-minute inhalation of Heroin 50 mg/mL, whereas male rats were only affected on the anti-nociception assay and only to an extent that failed to reach statistical significance. Overall, these patterns are more consistent with a dosing difference across sex rather than a sex difference per se; additional work with more expanded dosing conditions might further explicate this interpretation. Interestingly, when mg/kg equivalent doses were injected the males’ body temperature was consistently elevated whereas the females’ temperature was not. A previous study found no difference in the ED50 for heroin in a warm water tail-withdrawal test between male and female Sprague-Dawley rats and where sex differences were found the males were more sensitive. Nevertheless, female mice develop greater hypothermia than do males after a mg/kg equivalent injection of morphine . The initial suppression of activity, followed by a rebound of increased activity, is similar to that reported for injected morphine but differs somewhat from prior results for injected heroin, which appeared to show a monotonic effect of time where activity is highest at the first time-points observed after dosing and then declined steadily with time. The pattern of initial suppression followed by a rebound was observed in the 1.0 mg/kg, s.c. injection study, as well as in the 30-minute inhalation experiments, suggesting the difference is not due to route of administration. One possible difference is that some of those studies were conducted with rats in the light part of the cycle , however others reporting similar monotonic time courses were on a reverse cycle and were tested in the dark . A more consistent difference is that many studies of the effects of injected heroin used a non-housing, specialized photo cell cage with mesh or rod floors, unlike the plastic floored normal housing cages with a thin layer of bedding used here. It may be the case that the more familiar, housing-type environment facilitated expression of a more naturalistic response. There are a few necessary caveats; given the selected repeated measures experimental design it is always possible that a degree of plasticity, either sensitization or tolerance, may have developed. The counterbalanced testing order, however, minimizes the concern that this would have a systematic effect on any specific dosing conditions. Likewise, the animals had participated in prior studies involving exposure to THC, which might potentially produce cross tolerance, and because of that the animals were in the middle adult age range. It is entirely possible that responses in younger adults might differ.