The Effects Of Chronic Low-dose Administration Of Ketamine On Brain Functions In Adolescent Mice And Cynomolgus Monkeys

1IntroductionKetamine, a drug once used as an anesthetic for human and veterinary surgery, has increasingly become a mainstream’recreational’ drug. In previous researches, ketamine at a dosage which was much smaller than anesthetic dosage could brought a pleasant sensation of incredible intensively to ketamine users, like’A K-hole can be anything from going to hell and meeting Satan to going to heaven and meeting God’. In the United Kingdom alone, the prevalence rates of ketamine has rapidly increased2-fold from1999to2003among the nightclub goers, especially among the adolescents. While, Ketamine at the subanesthetic dose produced a series of positive and negative schizophrenic-like symptoms including perceptual alternations, visions, delusions, depersonalization and derealization in healthy individuals, and those impairments of abuse ketamine have aroused increasing attention from the international community and various countries. Recently, ketamine has become a controlled (Schedule Ⅲ of The Controlled Substances Act) substance in the USA and other countries.Ketamine is a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor, which regulates the action of excitatory aminoacids (EAAs) involving glutamate and asparte, and plays a crucial role in mechanisms of synaptic plasticity and neuronal learning. In the recent studies, many researches have validated the short-term effects of ketamine administration in healthy individuals, which induced impairments of cognitive function and psychological wellbeing, relatively little is known about the effects of long-term ketamine abuse. Obviously, the latter conforms to the status of sucking ketamine. Moreover, although rodents have been commonly used for drug administration studies, since the behavioral research of drug addiction and manifestation of working memory deficits, the monkey has emerged as a premier subject to model cognitive, behavioral and neuropsychopathological disorders, and to investigate novel drug treatments of the disease concerned cognitive dysfunction.’Cognition’ in nature, that is, the mental capacity to override or augment reflexive and habitual reactions in order to orchestrated behavior in accord with our intentions, is essential for what we recognize as intelligent behavior. In another word, the primary function of cognitive control is to extract the goal-relevant features of long-term memory programs for use in future circumstances. It has been validated that the prefrontal cortex (PFC) is centrally involved in this process, which requires two essential integrated components:a central executive and working memory buffers. Moreover, the proper interconnection of PFC provides a perfect infrastructure for synthesizing the various range of information needed for complex and extended behavior. Some evidences have showed that the dopaminergic system of PFC is particularly vulnerable to ketamine administration, but the effect of prolonged ketamine administration on PFC is still largely unknown. Currently, some studies performed on rodents have indicated that sustained exposure to ketamine produces programmed cell death in areas of the central nervous system (CNS) and increases the brain expression of pro-apoptosis factors belonging to the mitochondrial and the extrinsic apoptotic pathways. These studies open up the possibility that chronic exposure to ketamine in the CNS would interfere with learning and memory through a neurotoxic effect related to activation of apoptotic pathways. Because of the exacerbation of ketamine abuse in the world and the significance of PFC in cognitive control, we established the hypothesis:chronic ketamine administration of recreational dosage might produce permanent and irreversible deficits in brain functions due to neurotoxic effects involving the activation of apoptotic pathway in the prefrontal cortex.2Objectives2.1To observe body weight and neuromuscular strength, the sensitivity to a painful stimulus and spatial navigation learning and memory in ICR mice following chronic low dose of ketamine exposure.2.2To observe body weight and behavioral changes in adolescent cynomolgus monkeys following chronic low dose of ketamine exposure.2.3To investigate the effects of chronic low dose of ketamine on cell apoptosis of prefrontal cortex (PFC) in adolescence.2.4To investigate the effects of chronic low dose of ketamine on the brain functions in adolescence and its underlying mechanism.3Materials and methods3.1Animal model of ICR mice establishedNinety male ICR mice were randomly divided into3groups receiving1-,3-or6-month of daily intra-peritoneal injection of ketamine of30mg/kg or saline. In each group of30mice,20were injected with ketamine and10with saline as controls.3.2Body weight of ICR mice measurementThe weights of mice were recorded every week for adjustment of ketamine injection and monitoring of the well-being of the animals.3.3Behavioral studiesBehavioral tests were made for3days after ketamine or saline injection. Behavior tests including wire hang test, hot plate test and water maze test.3.4Apoptosis assay in ICR miceAfter behavioral tests, ICR mice were randomly chosen to be sacrificed. Brains were dissected and embedded in paraffin wax. Sections of PFC were cut coronally from paraffin blocks at4-μm thickness.TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) was used to assay apoptosis. Brain tissues sections were incubated with proteinase K, and TUNEL reaction mixture. Finally, the sections were developed with DAB kit. Apoptotic cells were assayed.3.5Western blotting analysis in ICR micePFC tissues were homogenized in cold lysis buffer. The sample was centrifuged at14,000rpm for30min at4℃, then the supernatant was collected and protein content was assayed colorimetrically.100μg total proteins were loaded onto a12% SDS-PAGE gel, electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were incubated with primary antibodies at4℃overnight following incubation of secondary antibodies for1h. The bound antibodies were then visualized and recorded using the ODYSSEY Infrared Imaging System (LI-COR Biosciences)3.6Animal model of cynomolgus monkeys establishedTwenty-four adolescent male cynomolgus monkeys were randomly divided into3groups of8monkeys:one group was given daily intravenous injection through the inner side of the lower limbs of1mg/kg body weight of ketamine in saline for1month, another group for6months and the last group were given daily saline injection as the control group also for6months.3.7Body weight of cynomolgus monkeys measurementEach monkey was weighed at the beginning of each week, so that the correct ketamine dose could be administered.3.8Behavioral observation in cynomolgus monkeys15-min video recordings were made for each monkey after ketamine or saline injection at1st,3rd,7th,14th,30th,31st,56th,112th,183rd,184th and185th day. Behavior observation including Move, Walk, Climb and Jump was conducted.3.9Apoptosis assay in cynomolgus monkeysTUNEL was also used to assay apoptosis. The protocol was same to the ICR mice’s.3.10Western blotting analysis in cynomolgus monkeysPFC tissues were homogenized in cold lysis buffer. The sample was centrifuged at14,000rpm for30min at4℃, then the supernatant was collected and protein content was assayed colorimetrically.30μg total proteins were loaded onto a12%SDS-PAGE gel, electrophoretically transferred to PVDF membrane. The membranes were incubated with primary antibodies at4℃overnight following incubation of secondary antibodies for1h. The membranes were developed using an enhanced ECL detection system. The intensity of bands was determined using the Image J14.0software.4Results 4.1Chronic low dose of ketamine administration inhibited the body weight increase in ICR mice following chronic ketamine exposureThe percentage increases in body weight for the ketamine and control groups at1,3and6months have increased significantly. The percentage increases were less in all ketamine groups than the percentage increases of the respective control groups. However, the differences in the percentage increases between ketamine groups and their respective control groups were not statistically significant.4.2Effects on behavioral tests of Chronic low dose of ketamine abuse in ICR miceAccording to the results of wire hang test and hot plate tests, after6months’ ketamine administration, the latency time and the total number of movements of the6-month ketamine group was significantly less than the6-month control group. However, there were no significant differences and consistent trends of changes in ketamine treated mice for1and3months as compared to their respective controls.According to the results of the water maze test, the escape latency time for the mice to locate and climb onto the platform showed no statistically significant differences between the1-,3-, and6-month ketamine groups as compared to their respective control groups. Although, the ketamine treated mice in different groups consistently used more time to climb onto the platform.4.3Effects on PFC apoptosis of Chronic low dose of ketamine abuse in ICR miceThere were no statistically significant differences of the TUNEL positive cell counts in the prefrontal cortex between the ketamine groups of1-,3-,6-month and their respective control groups. Although Western blot results showed consistently in all1-,3-, and6-month ketamine groups had higher Bax, lower Bcl-2, higher Bax/Bcl-2, and higher caspase-3levels in PFC than their respective control groups, all differences were not statistically significant.4.4Chronic low dose of ketamine administration inhibited the body weight increase in cynomolgus monkeys following chronic ketamine exposureBoth1-month and6-month ketamine and control groups increased their body weights about10%during the first4months. In the latter2months, the control group continued to gain about15%of body weight, while the ketamine group gained very little. However, there was no statistically significant difference between the groups.4.5Chronic low dose of ketamine abuse in adolescent cynomolgus monkeys depressed motor behaviorsFor behavior test, activities of Move, Walk, Climb and Jump of ketamine group exhibited obvious decreased tendency as time went by. With the exception of the results for Jump of1-and6-month ketamine groups versus controls (which were significantly different), results of other behavioral movements (Move, Climb, Walk and Total behavior) showed no significant differences between the ketamine and control groups. However, in the latter153days, results of Move, Walk, Jump and total behavior did show significant changes on different days. Furthermore, results of Total behavior movement showed significant differences both between different days and interactions between different days and control/ketamine groups. In addition, results of Move, Climb and Total behavior showed significant differences between the ketamine and control groups.4.6Effects on PFC apoptosis of Chronic low dose of ketamine abuse in adolescent cynomolgus monkeysTUNEL positive cells increased significantly (p<0.05) in the prefrontal cortex of the6-month ketamine group as compared with those of the control group; In Western blot assays, for the6-month ketamine group, Bax and caspase-3in the PFC both increased significantly (p<0.05) compared with the control group. Results of TUNEL positive cells, Bax, Bcl-2and caspase-3of the1-month ketamine group showed no significant differences as compared to the control group. Bcl-2decreased in the6-month ketamine group, but the difference as compared to the control group was not significant.5ConclusionKetamine at the usual recreational dose after6months of use could produce stable and persistent damages to the brain’s functions, decrease body weight gains. Mice showed significant deteriorations in the neuromuscular strength and nociception despite no significant apoptosis was found in prefrontal cortex. Monkeys showed locomotor activity decreased significantly, and ketamine treatment at recreational dose for6months might produce possibly permanent and irreversible deficits in brain function through the neurotoxic effect by activation of apoptotic pathway in PFC of adolescent monkeys. There might be regional differences in responses between different CNS regions and the detrimental effects of ketmaine on brains were not uniform across species, the primate animals were more sensitive to ketamine. These findings may have significant clinical implications in relation to chronic ketamine abuse and target therapeutic strategies.