Acepromazine and Chlorpromazine As Pharmaceutical-Grade Alternatives to Chlorprothixene for Pupillary Light Reflex Imaging in Mice

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Acepromazine and Chlorpromazine as Pharmaceutical-grade Alternatives to Chlorprothixene for Pupillary Light Reflex Imaging in Mice

Samantha S Eckley,1 Jason S Villano,1 Nora S Kuo,1 and Kwoon Y Wong2,*

Studies of visual responses in isoflurane-anesthetized mice often use the sedative chlorprothixene to decrease the amount of isoflurane used because excessive isoflurane could adversely affect light-evoked responses. However, data are not available to justify the use of this nonpharmaceutical-grade chemical. The current study tested whether pharmaceutical-grade sedatives would be appropriate alternatives for imaging pupillary light reflexes. Male 15-wk-old mice were injected intraperitoneally with 1 mg/kg chlorprothixene, 5 mg/kg acepromazine, 10 mg/kg chlorpromazine, or saline. After anesthetic induction, anesthesia maintenance used 0.5% and 1% isoflurane for sedative- and saline-injected mice, respectively. A photostimulus (16.0 log photons cm−2 s−1; 470 nm) was presented to the right eye for 20 min, during which the left eye was imaged for consensual pupillary constriction and involuntary pupil drift. Time to immobilization, loss of righting reflex, physiologic parameters, gain of righting reflex, and degree of recovery were assessed also. The sedative groups were statistically indistinguishable for all measures. By contrast, pupillary drift occurred far more often in saline-treated mice than in the sedative groups. Fur- thermore, saline-treated mice took longer to reach maximal pupil constriction than all sedative groups and had lower heart rates compared with chlorpromazine- and chlorprothixene-sedated mice. Full recovery (as defined by purposeful movement, response to tactile stimuli, and full alertness) was not regularly achieved in any sedative group. In conclusion, at the doses tested, acepromazine and chlorpromazine are suitable pharmaceutical-grade alternatives to chlorprothixene for pupil imaging and conceivably other in vivo photoresponse measurements; however, given the lack of full recovery, lower dosages should be investigated further for use in survival procedures.

Abbreviations: GORR, gain of righting reflex; LORR, loss of righting reflex; PLR, pupillary light reflex

DOI: 10.30802/AALAS-JAALAS-19-000094

Light-evoked pupillary constriction, also known as the pupil- conjunction with a sedative so that the level of isoflurane can be lary light reflex (PLR), is mediated by melanopsin-expressing, reduced considerably. A commonly used anesthetic approach intrinsically photosensitive retinal ganglion cells that drive for in vivo electrophysiologic studies of light-evoked responses various nonimage-forming visual responses as well as regu- is premedication with chlorprothixene, an antipsychotic drug of late conscious visual perception.21,24,26,34 Thus, PLR imaging the thioxanthene class, followed by isoflurane levels maintained is useful for assessing the properties and functions of these at or below 0.5%.11,27,39,44 Chlorprothixene is an antagonist at ganglion cells.18,20,34,35,49,52 PLR studies in mice sometimes use dopamine, serotonin, histamine, muscarinic acetylcholine, and anesthesia to immobilize the animals, but conventional veteri- α-adrenergic receptors.17,38 Chlorprothixene has been discontin- nary anesthetic regimens have presented various challenges. ued in the United States as a pharmaceutical-grade compound The inhalant anesthetic isoflurane has been shown to suppress but is still available in Europe.1,12,28 the PLR in humans.3,32 Meanwhile, xylazine, an α2 adrenergic According to The Guide for the Care and Use of Laboratory Ani- agonist typically used as a sedative and in combination with mals, pharmaceutical-grade chemicals “should…be used, when ketamine, can cause acute reversible lens opacities8 that interfere available, for all animal-related procedures” and “[t]he use of with pupillary imaging. nonpharmaceutical-grade chemicals or substances should be In PLR imaging studies, most of the stated complications may described and justified in the animal use protocol…”.30 The 2 be circumvented by hand-restraining unanesthetized mice,48 but primary concerns of using reagent-grade chemicals are steril- this method is infeasible for prolonged continuous imaging51 ity and efficacy. Sterility can be achieved through appropriate and could trigger anxiety and other autonomic responses that handling and filtration but is difficult to maintain when dosing alter the pupil size.5,7 Another strategy is to apply isoflurane in multiple animals. Efficacy is especially important for anesthetics and analgesics, because a decline in efficacy can immediately compromise animal welfare. The main advantage of using Received: 01 Jul 2019. Revision requested: 03 Sept 2019. Accepted: 09 Sept 2019. pharmaceutical-grade compounds is that they are formulated 1 2 Unit for Laboratory Animal Medicine, Department of Ophthalmology and Visual Sci- to account for such properties as purity, sterility, biocompat- ences, and Department of Molecular,Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan ibility, osmolality, and pH so that they are suitable for in vivo *Corresponding author. Email: [email protected]

197 Vol 59, No 2 Journal of the American Association for Laboratory Animal Science March 2020 use. In the United States, chlorprothixene is available only in suppression of involuntary pupillary drift during PLR imaging reagent-grade form, necessitating justification in the animal (see later section). Chlorprothixene powder was made to 8 mg/ protocol and extra precautions for use. To provide such justi- mL in 100% dimethylsulfoxide, and then diluted in saline to its fication, there should be clear benefits to using chloprothixene final concentration of 0.2 mg/mL and sterile-filtered through over other pharmaceutical-grade options, but no such benefits a 0.22-µm hydrophilic PVDF filter (Thermo Fisher Scientific, have been reported in the literature. Therefore, an appropriate Waltham, MA). Acepromazine and chlorpromazine liquid pharmaceutical-grade substitute needed to be identified. Two stocks were diluted with saline to their final concentrations of potential pharmaceutical-grade alternatives are acepromazine 1 and 2 mg/mL, respectively. The final concentrations of all and chlorpromazine, which are both phenothiazines with a drugs were determined such that the injection volume would similar chemical backbone structure and mechanism of action be the same, regardless of the drug. Each mouse was weighed to chlorprothixene. Acepromazine is an antipsychotic medica- shortly before dosing, and the injection volume was calculated tion, but its current use is mainly in animals as a sedative and according to the weight. antiemetic.41,47 Chlorpromazine has been used as an antipsy- After injection, mice were observed for 1 min for the chotic and antiemetic in humans.4,37 absence of adverse effects, such as hunching, jumping, uri- Here, we compared the use of chlorprothixene, acepromazine, nation, defecation, and increased activity. Anesthesia was and chlorpromazine as premedications when measuring murine then induced by using 4% isoflurane (VetOne) and a portable PLR. We hypothesized that acepromazine and chlorpromazine, anesthesia machine (Patterson Scientific, Waukesha, WI), and like chlorprothixene, would enable us to reduce the isoflurane times to recumbency and loss of righting reflex (LORR) were level needed to immobilize a mouse to 0.5% and that all 3 seda- determined. Time to recumbency was defined as the moment tives would yield quantitatively comparable PLR. In addition, when the animal’s body and head were in full contact with we examined PLR in nonsedative-treated mice that were im- the chamber floor. The onset of recumbency triggered the as- mobilized by using the lowest isoflurane level needed (1%), to sessment of LORR, which was determined by manually tilting learn whether this fairly low level would avoid suppressing the the chamber to place the animal on its back and assessing its PLR, thereby obviating the need for sedatives. ability to right itself within 10 s. When the mouse righted itself, then the next assessment was made when recumbency was Materials and Methods again achieved. After LORR was attained (at approximately Animals. All procedures were approved by the University 2.4 min after injection), the isoflurane level was lowered to of Michigan IACUC. Male 15-wk-old B6129SF2/J mice (The 1% until 5 min postinjection, when it was reduced further to Jackson Laboratory, Bar Harbor, ME) were group-housed in 0.5% for the sedative groups for the remainder of the study; IVC (Allentown, Allentown, NJ) on Pure-o’Cel bedding (The isoflurane was kept at 1% for mice injected with saline, to Andersons Lab Bedding, Maumee, OH) with unrestricted maintain immobilization. access to triple-filtered city water; we tested just one sex and Imaging. Pupil imaging was performed between 0900 and one age to minimize the number of animals required and the 1600. Mice were placed sternally on a recirculating water blan- number of variables. Animal rooms were maintained on a ket (K-Mod 100 Heat Therapy Pump, Baxter, Deerfield, IL) set 12:12-h light:dark cycle, relative humidity of 30% to 70%, and at 41 °C. The right pupil of each mouse was dilated by using temperature of 72 ± 2 °F (22.2 ± 1.1 °C). Health surveillance drops of 2.5% phenylephrine hydrochloride (Paragon BioTeck, program for SPF colonies included quarterly testing of dedicated Portland, OR) and 1% tropicamide (Akorn, Lake Forest, IL). soiled-bedding sentinel animals through fecal PCR analysis The left pupil was kept moist by using hypromellose lubricant or serology and PCR assay of exhaust plenum swabs for fur (Goniovisc, HUB Pharmaceuticals, Rancho Cucamonga, CA) mites. Surveillance results indicated that the mice were nega- and imaged continuously by using the EyeLink 1000 Plus eye tive for mouse rotavirus, mouse hepatitis virus, minute virus tracker (SR Research, Ottawa, Ontario, Canada). The mouse of mice, ectromelia virus, Theiler mouse encephalomyelitis was kept in darkness throughout imaging, except during virus, lymphocytic choriomeningitis virus, mouse adenovirus, photostimulation. Baseline imaging was performed for 2 min. −2 −1 mouse parvovirus, mouse polyomavirus, pneumonia virus of Then, a light stimulus (16.0 log photons cm s ; 470 nm) was mice, reovirus, Sendai virus, Mycoplasma pulmonis, pinworms applied selectively to the right eye for 20 min—this intensity, (Syphacia spp. and Aspicularis spp.), and fur mites (Myobia mus- wavelength, and duration are often used by the last author 51 culi, Myocoptes musculinis, and Radfordia affinis). to assess the sustainability of retinal photoresponses —and Sedatives and anesthesia. All mice were dark-adapted over- the left eye was imaged for consensual PLR and involuntary night prior to the experiment. All experimental procedures pupillary drift. The images captured by the eye tracker were prior to pupil imaging were conducted under mostly dim red transmitted in real time to another computer by using a frame light (<13.3 log photons cm−2 s−1), with brief exposures to dim grabber at 1 Hz, and pupil diameter was measured offline by white light (<40 lx) totaling approximately 5 min. Although using a LabVIEW-based image processing routine (Figure 1) Monitoring. Heart rate, blood oxygen saturation (SpO ), and these prior light exposures likely affected the PLR, all mice 2 were exposed to similar levels and durations of red and white rectal temperature were monitored approximately every 5 min light, and each mouse was allowed to dark-adapt for approxi- during anesthesia by using a commercial device (PhysioSuite, mately 8 min just before PLR imaging. A total of 40 mice were Kent Scientific, Torrington, CT). A rectal probe was inserted to used, which were randomly divided into groups of 10. Mice in monitor temperature, and a pulse oximeter sensor was placed each group were injected intraperitoneally with either 1 mg/ on one of the hindpaws to monitor blood oxygen saturation kg chlorprothixene (Sigma-Aldrich, St Louis, MO), 5 mg/kg and heart rate. In addition, toe pinch and tail pinch responses acepromazine (VetOne, Boise, ID), 10 mg/kg chlorpromazine were evaluated shortly before and shortly after imaging, to (Hikma Pharmaceuticals, Eatontown, NJ), or 0.9% saline (Hos- assess anesthetic depth. All measurements were performed by pira, Lake Forest, IL). These sedative doses were the maximums observers (SE, NK) blinded to the sedative used, although the recommended by the Unit for Laboratory Animal Medicine at saline group precluded blinding due to the increased level of the University of Michigan, and we chose them to maximize the isoflurane required.

198 Anesthetic regimens for pupil imaging in mice

Figure 1. The LabVIEW-based program for measuring pupil diameter. First, at least 3 points are selected to outline the pupil (left), and the circle that best fits the points is generated automatically (right). The red value is the area of this circle, and the white value is its diameter; both values are in arbitrary units.

Recovery. Mice were recovered on a microwavable heating showed some degree of pupillary drift (Figure 2 A) This drift bag and observed for time to gain of righting reflex (GORR) had no consistent pattern in location or timing but did hinder and degree of recovery. The GORR was determined by placing diameter measurement when the pupil moved under the eye- mice in dorsal recumbency and assessing when they flipped to lids. In all anesthetic regimens, the pupils constricted on light their ventral side. Recovery was categorized as minimal, partial, stimulation of the contralateral eye (Figure 2 B). Baseline pupil or full, according to the level of alertness, response to tactile diameter, minimum pupil diameter, and constriction amplitude stimulation and spontaneous movement. Tactile stimulation was near the end of the 20-min illumination did not differ among performed by periodically lightly stroking the mouse’s body the 4 groups of mice (Figure 2 C). However, the saline-treated and observing for resultant movement. Minimal recovery was mice took approximately twice as long to reach maximal con- defined as a sedate mouse with a slow response to tactile stimu- striction than the mice treated with acepromazine (P = 0.0029), lation and no spontaneous movement. Partial recovery was chlorpromazine (P = 0.0090), or chlorprothixene (P = 0.0074; defined as a sedated mouse with a more immediate response Figure 2 C). to tactile stimulation, and intermittent voluntary purposeful Induction and recovery. Induction was rapid with no sig- movement. For full recovery, the mouse was alert with normal nificant intergroup difference in time to recumbency or time to voluntary movement. Mice were observed until they fully recov- LORR (Table 1) No adverse effects were noted in any group. ered or for at least 1 h after the end of anesthesia. The recovery Recovery was categorized as detailed in Materials and Meth- status of the mouse after the 1-h observation determined their ods. Two mice in the acepromazine group, one mouse in the recovery categorization. Mice were euthanized by the end of chlorpromazine group, and 3 mice in the chlorprothixene group the day by using CO2 followed by cervical dislocation. achieved only minimal recovery within 60 min of the end of Statistical analyses. Statistical analyses were performed by anesthesia. None of the mice that received acepromazine or using Prism 7.0 (GraphPad, La Jolla, CA) or R version 3.6.0 chlorpromazine achieved full recovery, whereas 3 mice in the (https://www.r-project.org/). Data were assessed for Gaussian chlorprothixene group and all of the mice in the saline group distribution according to the D’Agostino and Pearson omnibus did (Table 1). The time to GORR was not significantly different normality test as well as by visual inspection. Comparison of among the groups. Time to partial or full recovery is reported groups for induction, recovery, and pupillary parameters used only for mice that reached that level of recovery. Neither time to either one-way ANOVA followed by the Tukey multiple-compar- partial recovery nor time to full recovery differed significantly isons test for parametric data or Kruskal–Wallis analysis followed among the treatment groups (Table 1). by the Dunn multiple comparisons test for nonparametric data. Anesthetic parameters. Heart rate, blood oxygen saturation, To assess the sedative effect on physiologic measurement time and temperature remained relatively steady throughout anes- courses, a linear mixed-effect model9 was used, with a sedative thesia (Figure 3 A through C). Heart rate measurements from as a fixed effect and mouse as a random effect. Models including one saline-treated mouse were excluded from analysis due to time did not influence the results and therefore were not included. poor readings. The heart rate time course was significantly lower Regression analysis was performed in R software by using the for saline-treated mice than for those given chlorpromazine (P lme4 package2 with posthoc testing using least-squares means = 0.0257) or chlorprothixene (P = 0.0212; Figure 3 A). For blood and the Tukey test in the lsmeans package.33 P values less than oxygen saturation, one chlorpromazine-treated mouse and one 0.05 were considered statistically significant. saline-treated mouse were excluded from analysis due to poor readings. Blood oxygen saturation was maintained above 90% Results for all groups, with no significant differences (Figure 3 B). Tem- Light-evoked pupillary responses. In the sedative groups, perature in saline-treated mice seemed to trend higher than for pupils remained relatively steady throughout PLR imaging the sedative groups, but there were no statistically significant and were readily analyzed. However, 7 mice in the saline group differences (Figure 3 C).

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Figure 2. Light-evoked pupillary responses in mice (n = 10) that received premedication with acepromazine, chlorpromazine, chlorprothixene, or saline followed by isoflurane anesthesia. (A) Pupillary drift examples from one saline-treated mouse, imaged at 4 time points while the mouse was kept in darkness. The red curve in each image outlines the pupil. (B) Pupillary diameter (mean ± SEM) normalized to baseline over the imaging period for each treatment group. Left to right: acepromazine, chlorpromazine, chlorprothixene, and saline. (C) Left to right: baseline pupil diameter, time to maximal constriction, minimum pupil diameter as a percentage of the baseline diameter, and constriction at 20 min as a percentage of the maximal constriction (mean ± SEM). The number above each column indicates the n value for that measurement. Although there were 10 mice in each treatment group, some n values are less than 10 because some pupil images could not be analyzed due to excessive glare, pupillary drift, or eye closure. †, P < 0.01.

Table 1. Induction and recovery parameters in mice (n = 10) that received premedication with acepromazine, chlorpromazine, chlorprothixene, or saline. All times from injection. Values are mean ± SEM. Induction Recovery Time to Categorization recumbency Time to Time to Time to partial Time to full (min) LORR (min) Minimal Partial Full GORR (min) recovery (min) recovery (min) Acepromazine 2.15 ± 0.09 2.34 ± 0.11 2 8 0 47.22 ± 2.38 61.58 ± 3.69 NA Chlorpromazine 2.16 ± 0.04 2.40 ± 0.08 1 9 0 46.60 ± 2.19 62.94 ± 4.25 NA Chlorprothixene 2.06 ± 0.06 2.37 ± 0.11 3 4 3 45.17 ± 1.93 52.06 ± 4.93 51.69 ± 2.74 Saline 2.15 ± 0.08 2.23 ± 0.10 0 0 10 50.04 ± 2.44 NA 50.95 ± 2.61 NA, not applicable; GORR, gain of righting reflex; LORR, loss of righting reflex

The toe and tail pinch responses were lost inconsistently dur- Discussion ing anesthesia (Table 2) The tail pinch response was absent more This study investigated different premedication regimens, at frequently than the toe pinch response. In the acepromazine the highest recommended sedative doses, with regard to PLR group, only 3 mice lost the toe pinch response at some point and influence on anesthetic induction, physiologic parameters, during anesthesia. In all other groups, a majority of mice lost and recovery. Numerous studies have used the nonpharmaceu- the toe and tail pinch responses for at least one measurement. tical-grade drug chlorprothixene (for example, references 11, Very few mice (0 to 3 mice per treatment group) had absent 27, 39, and 44) to decrease the level of isoflurane, but justifica- responses during the entire anesthetic period (Table 2). tion is sparse. We assessed acepromazine and chlorpromazine

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not observe these specific phenomena, but they indicate the possibility of variable involuntary eye movements depending on the anesthetic regimen. The slower pupillary constriction in the saline group may be explained by the higher level of isoflurane (1% compared with 0.5%) needed to immobilize these mice for imaging. Several anesthetics have been shown to attenuate the PLR in various species.3,15,22,23,29-32,45 Dogs had significantly reduced constriction velocity and peak amplitude during anesthesia with ketamine–xylazine–atropine compared with preanesthesia.31 The inhaled anesthetics isoflurane, sevoflurane, and enflurane decrease PLR amplitudes in both mice and humans.3,29,32,45 In our current study, the only physiologic parameter to show an intergroup difference was heart rate, with the saline group significantly lower than the chlorpromazine and chlorprothixene groups. The isoflurane level cannot solely account for this difference. In mice, heart rate was actually higher when the isoflurane level was increased from 1% to 1.5% or 2%.10 Rats showed dose-dependent decreases in heart rate with increased isoflurane concentrations, but not until the isoflurane level was 2%.50 Rather, the difference in heart rates may be secondary to the profound vasodilation that can be caused by chlorprothixene and similar chemicals.13,16,41 Presumably, vasodilation results in lower mean arterial pressure, which then triggers a compensa- tory increase in heart rate. The toe pinch and tail pinch responses in the mice were not consistently absent during anesthesia. At most, only one mouse in each group lost both responses when checked during the anesthetic period. In general, the responses were more likely to Figure 3. Physiologic parameters (mean ± SEM) measured over time in mice (n = 9 or 10) that received premedication with acepromazine, be absent earlier in the anesthetic period, with the mouse being chlorpromazine, chlorprothixene, or saline followed by isoflurane an- more responsive at the end of imaging. These results indicate esthesia. For graphical display, values were binned in 5-min intervals. that the regimens investigated are suitable only for nonpainful (A) Heart rate. (B) Oxygen saturation. (C) Rectal temperature. *, P < procedures. 0.05. The recovery of the mice was substantially sedative-dependent. All saline-injected mice recovered quickly as expected, as possible pharmaceutical-grade options to chlorprothixene. whereas all sedative-treated mice had poorer and longer re- We found that the saline group had significantly slower PLR coveries. The 4 groups did not differ with regard to GORR, kinetics compared with the sedative groups, suggesting that 1% but the sedative-treated mice had profoundly greater levels of isoflurane probably impedes the murine PLR to some degree sedation than the saline-treated mice. The dosages, especially (discussed later), but other pupillary measures were statistically of acepromazine and chlorpromazine, were at the high end similar across all 4 groups. In addition, anesthetic induction, toe of reported dosages in the literature14,19,27,36,40,42 to maximize and tail pinch responses, oxygen saturation, temperature, and the suppression of involuntary pupil drift and to ensure that GORR did not differ among the groups. By contrast, the heart the isoflurane level could be lowered to 0.5%. However, this rate was significantly lower for saline than for chlorpromazine high dosage likely also produced the prolonged sedation ob- and chlorprothixene. Moreover, the level of recovery differed, served during recovery. If these sedatives are used for survival most notably between the saline and sedative groups. These procedures, we recommend lowering the dosages to improve results suggest that for the doses tested, the sedative groups recovery. perform similarly and are appropriate for measuring PLR; This study had several limitations. First, anesthetic induction therefore, acepromazine and chlorpromazine are effective began only 1 min after injection, so the sedatives likely had not pharmaceutical-grade substitutes to chlorprothixene. taken full effect before assessment of time to recumbency and The largest detriment to using 1% isoflurane without a time to LORR began. Sedative effects were probably more com- sedative was the excessive pupillary drift that occurred, which plete for the subsequent assessments, because it took at least 10 precluded pupil diameter measurement at various time points. min to prep the mouse for the imaging procedure, and all mice The drift was not consistent with regard to timing or location. could be immobilized at 0.5% isoflurane throughout the pupil Some mice had continuous drift throughout the imaging in- imaging. Second, induction used a fairly high isoflurane level terval, whereas others had intermittent drift. Pupil position in (4%), which could have obscured sedative effects and reduced animals is known to be affected by anesthetic depth, which is intergroup differences in induction times. Third, during pupil 25 often used as an adjunct measure. The absence of pupil drift imaging, the need for 1% isoflurane in the saline group reduced in the sedative groups might be due to an increased depth of the level of blinding in the study, although the sedative groups anesthesia. Involuntary pupillary movement has been reported remained blinded from the observers. Fourth, we used only with other anesthetic regimens. For instance, nystagmic move- one dosage for each sedative. A dose–response characterization 43 ment has been observed in ketamine-anesthetized rats. Pupil would have helped to determine the minimal effective dosage size in rats has been reported to show very slow periodic fluc- but also would have substantially increased the number of mice 6,46 tuations during isoflurane and urethane anesthesia. We did needed to complete these studies. For our purpose of identifying

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Table 2. Toe pinch and tail pinch responses in mice (n = 10) that received premedication with acepromazine, chlorpromazine, chlorprothixene, or saline followed by isoflurane anesthesia. No. (%) of mice with absent toe pinch response No. (%) of mice with absent tail pinch response Absent at any point Absent at any point Absent for during anesthesia Absent for entire anesthesia during anesthesia entire anesthesia Acepromazine 3 (30%) 1 (10%) 9 (90%) 3 (30%) Chlorpromazine 6 (60%) 1 (10%) 9 (90%) 2 (20%) Chlorprothixene 7 (70%) 0 (0%) 8 (80%) 1 (10%) Saline 8 (80%) 1 (10%) 7 (70%) 1 (10%) appropriate sedatives to suppress pupillary drift and reduce 4. Bhargava KP, Chandra O. 1963. Anti-Emetic activity of pheno- the isoflurane level to 0.5%, a single maximal concentration thiazines in relation to their chemical structure. Br J Pharmacol was sufficient, but may not be for other applications. Fifth, we Chemother 21:436–440. https://doi.org/10.1111/j.1476-5381.1963. used noninvasive methods for monitoring, which proved to be tb02011.x. 5. Bitsios P, Szabadi E, Bradshaw CM. 1996. The inhibition of the challenging at times. Specifically, in a few mice and for unknown pupillary light reflex by the threat of an electric shock: a potential reasons, the pulse oximeter had difficulty reading heart rate or laboratory model of human anxiety. J Psychopharmacol 10:279– blood oxygen saturation. In addition, we did not assess blood 287. https://doi.org/10.1177/026988119601000404. pressure, which would have been informative given the known 6. Blasiak T, Zawadzki A, Lewandowski MH. 2013. Infra-slow effects of the sedatives and observed differences in heart rate oscillation (ISO) of the pupil size of urethane-anaesthetised compared with the saline group. Sixth, to minimize the number rats. PLoS One 8:1–12. https://doi.org/10.1371/journal. of variables, we used only one mouse strain of one sex and a pone.0062430. specific age, so the findings might not be applicable to other ages 7. Bushnell M, Umino Y, Solessio E. 2016. A system to measure the pupil response to steady lights in freely behaving mice. or to female mice. We chose B6129SF2/J mice because they are J Neurosci Methods 273:74–85. https://doi.org/10.1016/j. used routinely in the last author’s lab , but genetic variability is jneumeth.2016.08.001.Erratum in J Neurosci Methods 2018. 299: somewhat high in these hybrid mice, which could potentially 64.https://doi.org/10.1016/j.jneumeth.2017.08.011. decrease intergroup statistical differences. 8. Calderone L, Grimes P, Shalev M. 1986. Acute reversible cataract In conclusion, we found that all of the sedatives tested pro- induced by xylazine and by ketamine-xylazine anesthesia in rats duced similar effects on PLR and anesthesia, at least for the and mice. Exp Eye Res 42:331–337. https://doi.org/10.1016/0014- doses tested and for 15-wk-old male mice. Saline-treated mice 4835(86)90026-6. anesthetized at an increased isoflurane level had slower PLR 9. Cnaan A, Laird NM, Slasor P. 1997. Using the general linear mixed model to analyse unbalanced repeated measures and longitudinal and greater pupillary drift. All sedatives in combination with data. Stat Med 16:2349–2380. https://doi.org/10.1002/(SICI)1097- isoflurane prolonged recovery and did not produce a surgical 0258(19971030)16:20<2349::AID-SIM667>3.0.CO;2-E. plane of anesthesia, indicating these dosages may be used for 10. Constantinides C, Mean R, Janssen BJ. 2011. Effects of isoflurane nonpainful, terminal procedures. Taken together, these results anesthesia on the cardiovascular function of the C57BL/6 mouse. demonstrate that pharmaceutical-grade acepromazine and ILAR J 52:e21–e31. chlorpromazine are appropriate alternatives to chlorprothixene 11. Dasilva M, Storchi R, Davis KE, Grieve KL, Lucas RJ. 2016. for use in mouse PLR studies. Although we examined PLR only, Melanopsin supports irradiance-driven changes in maintained acepromazine and chlorpromazine potentially could be suitable activity in the superior colliculus of the mouse. Eur J Neurosci 44:2314–2323. https://doi.org/10.1111/ejn.13336. substitutes for chlorprothixene in studies of other photore- 12. Deurell M, Weischer M, Pagsberg AK, Labianca J. 2008. The use sponses, and we recommend researchers consider trying these of antipsychotic medication in child and adolescent psychiatric agents in their experiments. treatment in Denmark. A cross-sectional survey. Nord J Psychiatry 62:472–480. https://doi.org/10.1080/08039480801985096. Acknowledgments 13. Dobkin AB, Byles PH, Lee PKY, Israel JS. 1963. 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