Brimonidine is clinically used in the treatment of glaucoma and facial congestion. No previous study has investigated the induction or maintenance of general anesthesia with brimonidine; however, a report of brimonidine administration before general anesthesia in animals, such as intraperitoneal injection of 30µg/kg brimonidine, diminished the sympathetic nerve effect of ketamine general anesthesia in rats [1]. In addition, there are many reports regarding the use of brimonidine for local anesthesia studies. For example, preoperative use of brimonidine eye drops to prevent an increase in intraocular pressure also improved the local analgesic effects of ophthalmic operations, such as cataract phacoemulsification [2]. Moreover, 0.33% brimonidine gel combined with lidocaine cream prolonged the local anesthesia time in skin surgery [3]. Inhibition of thermal nociception in mice following epidural administration of brimonidine had an analgesic ED50 of 0.37 nM, and the combination of brimonidine with an epidural, such as opioids, can lead to synergistic analgesia [4]. Epidural administration of brimonidine also had analgesic effects in a rat model of chronic neuropathic pain [5, 6].
Many clinical reports indicate that local and systemic administration of brimonidine can cause side effects in the central nervous system, resulting in nervous system symptoms such as sedation, lethargy, and even death [7]. Following intravenous injection of 1 mg/kg brimonidine, the rats stopped moving, their pupils dilated (different from those of primates and rabbits, which may be related to species specificity), and they calmed down. After intravenous injection of 2 mg/kg brimonidine, the rats breathed slowly and quietly, and the dose did not reach the level of subliminal anesthesia [8]. Toxicity testing revealed that a large dose (5 mg/kg) of oral brimonidine elicited short-term hypnotic and sedative effects in rabbits, which also exhibited disappearance of nerve responses and reduced muscle tension [9]; however, the effect of general anesthesia was not studied in depth.
This study confirmed that brimonidine has good hypnotic, sedative, and analgesic effects on mice and rabbits. This result is related to the α2 receptor agonist action of brimonidine.
Human and mammalian α2-adrenergic receptors belong to a family of seven-transmembrane G protein-coupled receptors, which can transfer Gi/Go protein and have the following effects: inhibition of adenylate cyclase and protein kinase A; activation of protein kinase C; activation of potassium channels to hyperpolarize cells and produce postsynaptic inhibition; and inhibition of calcium influx and neurotransmitter release at nerve endings, leading to presynaptic inhibition [10]. There are three α2 receptor subtypes in humans, primates, pig, and rabbit (α2a, α2b, and α2c), which produce different effects. The Ki of human α2a, α2b, and α2c receptors for brimonidine are 3.7, 512, and 120 nM, respectively [11].
The locus coeruleus brainstem is an important functional area in the central nervous system that regulates arousal. Activation of α2a receptors hyperpolarizes nerve cells in the locus coeruleus, which reduces the release of norepinephrine and inhibition of γ-GABA neurons in the ventrolateral anterior cross nucleus of the hypothalamus to induce sedative hypnotic effects. The ED50 of brimonidine for inducing potassium influx in rat locus coeruleus neurons in vitro is 4.6 * 10− 8 M, while 10− 6 M brimonidine can induce the maximum current [12]. The hypnotic mechanism of brimonidine is similar to that observed in normal non-eye-moving sleep. During observations of the hypnosis of rabbits, we found that the eyeball moves back and forth rapidly and slightly, and it is speculated that the rabbits entered a state similar to sleep.
The hypnotic assessment method was based on prolongation of sleep induced by pentobarbital, a classic pharmacological method for screening sedative hypnotic agents [13, 14]. The hypnotic effect was evaluated by sleep time duration induced by a subhypnotic dose of pentobarbitone. Brimonidine showed hypnotic activity that increased sleeping time induced by pentobarbitone.
In this study, we found that the ED50 of mouse hypnosis (78 mg/kg, intraperitoneal administration) and rabbit hypnosis(5.20 mg/kg, intravenous injection; 8.8 mg/kg, intramuscular injection;8.7 mg/kg, intrarectal injection), and sleeping time of mice (4.8 ± 0.5 min, intraperitoneal injection) and rabbits (128.1 ± 16.9 min, intravenous injection; 19.0 ± 11.3 min, intramuscular injection ;53.6 ± 27.9 min,intrarectal injection). The observed difference in hypnotic doses of brimonidine in mice and rabbits may be related to the difference in species. Thus, if brimonidine is developed as an anesthetic for experimental animals, it is more suitable to be used alone in rabbits than mice.
The α2 receptor agonist can exert analgesic and noxious stimulation inhibitory effects in peripheral nerves, spinal cord, and upper spinal cord [15]. The α2a subtype is contained in the spinal dorsal horn and primary sensory neurons, whereby adrenergic receptor agonists bind this receptor to trigger postsynaptic inhibition of secondary afferent neurons and presynaptic inhibition of primary sensory neurons, thus playing a major role in inhibiting pain signals [16]. The spread of chronic inflammation and neuropathic pain can also be inhibited by the anti-sympathetic nerve pathway. Activation of the α2c receptor inhibits the activity of nociceptive neurons in the dorsal root ganglion and spinal dorsal horn, but also reduces the release of endogenous opioids, leading to complex analgesic effects [17].
The acetic acid-induced writhing reflex mouse model is a widely accepted, simple, sensitive, and effective pain model for evaluating peripherally acting analgesics. The pain caused by acetic acid liberates endogenous substance to elicit peripheral- and central-mediated analgesic actions [18]. In the writhing test of mice, the analgesic effect of brimonidine at a dose of 0.016 mg/kg was obvious—this dose is 4,800 times lower than the hypnotic ED50. Compared with 0.016 mg/kg brimonidine, 0.08 mg/kg brimonidine had a sedative effect and an increased analgesic effect. A dose of 0.4 mg/kg brimonidine decreased the activity of mice, and no acetic acid-induced peritoneal pain reaction was observed. These results indicate that brimonidine significantly reduced the number of writhes in a dose-dependent manner compared with the negative control.
The hypnotic effect of brimonidine was more obvious in rabbits than in mice. Our experiments found that intravenous administration of 4.5 mg/mL brimonidine could make the righting response disappear. During this period, the limbs could be placed at will, but pinching the limbs could cause muscles of the limbs to contract. Although α2 receptor agonists can theoretically cause presynaptic inhibition of motor neurons and play a certain role in muscle relaxation [19], muscle relaxation is mainly caused by higher central inhibition. The limb muscle contraction caused by the clamp proves that the sedative effect of the above dose was good, but it did not achieve a hypnotic effect. The subthreshold dose of brimonidine in rabbits was far less than the anesthetic effective dose (not obvious in mice), but a good sedative effect at the low dose still caused some interference in hypnotic dose experiments in rabbits. Therefore, in the hypnotic half-effective-amount experiment of rabbits under intravenous intramuscular anesthesia, the acupuncture response rather than the righting response was specifically selected as the test criterion for sleep. The dose of brimonidine that inhibited the acupuncture response maintained the depth of anesthesia for simple surgery in small animals, while the low dose of brimonidine was suitable for pre-physical sedation.
Regarding the hypnotic effect of brimonidine on rabbits, pupil contraction was observed and the time of pupil contraction was very similar to the time of sedative action. The degree of pupil contraction can be used to roughly evaluate the agonistic performance of highly selective α2 receptor agonists on central α2 receptors of rabbits, monkeys, and other animals [20]. α2 receptor agonists stimulate α2 receptors in the postsynaptic membrane of neurons in the oculomotor nucleus, thereby inhibiting the release of noradrenaline from sympathetic nerve endings. This effect makes the contraction of pupil sphincter more dominant than the contraction of pupil opening muscle, resulting in pupil contraction. Brimonidine is insensitive to α1 receptors. When the local concentration reaches 10− 4 M, contractile force of the psoas major muscle is only 8%, which is not enough to offset central mydriasis [19]. It was also found that the conjunctiva of rabbits and mice turned white after administration of brimonidine, because brimonidine caused the contraction of anterior small blood vessels by activating α2a receptors of small blood vessels; however, brimonidine did not easily cause contraction of retinal blood vessels [21]. A local brimonidine concentration of 10− 4 M had no significant influence on retinal blood supply or function [22].
Although brimonidine has hypnotic and analgesic effects because it indirectly inhibits the ascending activation system of the reticular structure through the locus coeruleus, it does not block the awakening mechanism while accompanying sleep [23]. We found that during the hypnotic process elicited by injected administration of brimonidine to rabbits, they would be awakened in a short time by changing their body position and pulling their limbs hard. Therefore, brimonidine alone is only suitable for minor operations that do not involve frequent physical renewal. In addition, animals urinated during anesthesia, which might be due to the high dose of brimonidine acting on α2 receptors in the paraventricular nucleus of the hypothalamus, thereby inhibiting the release of antidiuretic hormone to cause an increase in urine output. Simultaneously, brimonidine inhibited norepinephrine release and contraction of the bladder sphincter, leading animals to urinate. Dexmedetomidine had a similar effect on diuresis in clinical applications [24]. This is disadvantageous for simple anesthesia of animals without catheter insertion because sudden micturition can affect disinfection of the surgical area.
After a high dose of brimonidine was intravenously administered, the hypnotic effect was not enhanced, but weakened. During the induction of general anesthesia, high-dose α2 receptor agonists might activates dopamine neurons in the ventral tegmental area and increases dopamine concentrations in the related forebrain projection areas [25] .During anesthesia, extrapyramidal symptoms appeared in some rabbits, and the extrapyramidal reaction might further weaken the inhibitory effect of drugs on central adrenal energy and affect the anesthetic effect.
To overcome these shortcomings and solve potential problems with future application, the combination of brimonidine with other anesthetics is a good method. In clinical anesthesia, dexmedetomidine can strengthen the efficacy of other central sedative drugs and analgesic drugs, and reduce the dosage of other drugs, thus reducing toxic and side effects. The present study found that brimonidine, a highly selective α2 receptor agonist with an imidazoline ring, has a similar effect. The results of experiments examining the hypnotic effect of brimonidine on increasing the threshold dose of pentobarbital confirmed the hypnotic effect of brimonidine. Moreover, these results revealed that 10 mg/kg brimonidine combined with 30 mg/kg pentobarbital hypnotized all mice for more than 2 h after intraperitoneal injection, without any death phenomenon, which is beneficial for long-term operations of mice. However, α2 receptor agonists such as brimonidine can lead to hypothermia [26], meaning small animals such as mice and rats may be injured or die. Therefore, in this study, mice were incubated to maintain a normal physiological body temperature.
In addition to pentobarbital, a combination of brimonidine and chloral hydrate was also found to have a good synergistic effect. When brimonidine and chloral hydrate were used together, the doses of the two drugs in the low-dose group were only one-tenth of the original single dose. In addition, the low-dose group exhibited rapid onset of anesthesia, a stable anesthesia effect, and convenient supplement of anesthetics. Although the dosage of the high-dose group was double that of the low-dose group, the anesthetic effect was similar. Thus, combining brimonidine and chloral hydrate has the potential advantages of stable effects and high cost performance for animal anesthesia.
Chloral hydrate, a central inhibitor, is seldom used alone at present. Mainly used for sedation and auxiliary anesthesia, chloral hydrate can quickly induce drowsiness and sedation, and has the advantages of fast anesthesia induction and recovery, high cost performance, and reliable sources. However, due to its limited analgesic effect, the dose required for anesthesia is close to the toxic amount and the safety range is small. Excessive chloral hydrate can inhibit myocardial contraction of animals and shorten the refractory period, leading to respiratory depression and death [27].
Chloral hydrate obviously accelerated the induction time of brimonidine anesthesia, and the application of brimonidine overcame the disadvantage of unsatisfactory anesthesia depth and muscle relaxation effects of chloral hydrate alone. Indeed, the reduced mixture had good anesthesia depth and muscle relaxation effects. The synergistic mechanism involves chloral hydrate further enhancing the analgesic effect of α2 receptor agonists by inhibiting the synaptic function of γ-aminobutyric acid (GABA) neurotransmission, whereas the inhibitory effects of the two drugs on respiratory and cardiovascular diseases may not exhibit synergy [28]. In addition, the combined use of drugs also reduces their respective doses, which is beneficial for both the effectiveness and safety of combined anesthesia.
The synergistic effect of brimonidine and chloral hydrate was compared with the previously reported formula of xylazine and chloral hydrate [29]. Xylazine, a non-imidazoline cyclic middle-selective a2 receptor agonist only used in veterinary medicine, is cheap and can be used for animal anesthesia with sedative drugs such as chloral hydrate. After intramuscular injection of xylazine alone, the anesthetic effect is uncertain and additional anesthesia is prone to accidents. Spasm and tremor are obvious during the induced excitement period; the maintenance period of anesthesia is short; and the decrease of muscle tension is often not significant. Moreover, xylazine readily influences cardiovascular functions and causes arrhythmia.
Experiments revealed that the individual effects of combined anesthesia with xylazine and chloral hydrate varied significantly, and one animal died after anesthesia, which was related to the limited drug consumption and high self-risk of xylazine and chloral hydrate in combination. In addition, a previous report indicated that chloral hydrate combined with xylazole, another α2 receptor agonist with low receptor selectivity, can cause obvious arrhythmia in anesthetized animals [30]. In contrast, brimonidine has higher receptor selectivity and lower cardiovascular side effects than low-selectivity α2 receptor agonists [31].
Two rabbits in the chloral hydrate combined with xylazine group were found to have ear vein inflammation a few days later, which may have been caused by the chloral hydrate solution. No ear vein inflammation was observed in high-dose or low-dose groups administered chloral hydrate combined with brimonidine, which may be related to the low dosage of the combined medicine.
In terms of safety, the therapeutic index of intraperitoneal brimonidine administration in mice was 5.0,while the therapeutic index of intravenous brimonidine in rabbits was 28.0, indicating that the safety range of brimonidine was wider in rabbits than mice. In this study, brimonidine was administered in combination with pentobarbital and chloral hydrate, and no deaths occurred.
There have been many reports on the safety of brimonidine administered to animals at high doses; for example, oral administration of brimonidine to rats for up to 1 year (2.5 mg/kg/day) did not cause organ toxicity [8]. In a study of reproductive toxicity, oral administration of 5 mg/kg brimonidine to pregnant rabbits caused abortion in individual rabbits, and had no teratogenic effect on offspring [9]. According to pharmacokinetic studies [32], brimonidine is metabolized in the liver and excreted in urine, with a half-life for blood elimination of 3 h and 29% binding rate of plasma protein. Such experimental conclusions have reference value for the use of brimonidine.
This sleeping time of rabbits in the 10-mg/kg intramuscular anesthesia group was shorter than observed in the 10-mg/kg intrarectal anesthesia group (Fiture2;P < 0.05). Use of brimonidine in clinical eye drops as a drug for glaucoma yielded high local absorption and no local tissue irritation [32] because it has proper physical properties (LogP = 0.83, PKa = 7.78), which makes brimonidine a potential sedative and anesthetic for mucosal administration ,when dexmedetomidine showed low bioavailability.
In future research, the safety of brimonidine for general anesthesia should be further investigated, such as monitoring of vital signs during anesthesia, investigating possible impacts on blood biochemistry and other indicators, and further confirming the efficacy of brimonidine combined with other anesthetics.