The Effects Of Chronic Low Dose Of Ketamine Administration On Brain Function In An Adolescent Primate Model

Studies indicate that developing brain is sensitive to drug abuse, in which damage may induce inreversible changes. Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that was abused rapidly recent years, with profound socioeconomic and health impacts. It was reported that even exposed little dose of ketamine to infant or adolescence can trigger widespread apoptotic neurodegeneration in brain tissue. Moreover, adolescence is a biologically critical period in neurodevelopment where reorganization, synaptic remodeling and receptor pruning occur in the dopaminergic system. Drug abuse can influence transmission of dopaminergic neurons, and then induce changes of growth and sensibility of reward system.Ketamine abuse can induce hallucinations, which is regarded as the main reason of drug addicts. Lots of reports demonstrated that ketamine abuser showed up objection of social ability, behavior and personality changes, damage of memory and organs, and especial evident damage of central nervous system. Present studies indicate that ketamine abuse can induce neuron apoptosis in many encephalic regions, including cerebral cortex, spinal cord, thalamus, hippocampus cingulate gyrus cortex and so on.Ketamine induces hypofunction of NMDAR including excessive glutamate release through decreased activity of GABAergic interneurons, and subsequent exciteds cortex which may induce psychosis-like behavior and cognitive anomalies. Previous studies have demonstrated that acute ketamine administration mainly influenced prefrontal lobe and periphery cortex. There are evidently increased levels of glutamic acid and dopamine in these different encephalic regions. However, the mechanism of chronic ketamine administration on prefrontal lobe, dopamine neurons is still unclear. The dopaminergic neurons play an important part in the reward system, in which glutamatergic-dopaminergic pathway interaction within the mesolimbic dopamine system are thought to contribute to addiction-related behavior in ketamine abuse. Functional magnetic resonance imaging (fMRI) about animal experiments showed that drug abuse enhanced activity of dopamine system in the mesolimbic, especially in striatum, robustly disrupting sensorimotor gating. With the emerging of new abused drugs successively, the modes of action and target areas of them are specificity differently. At present, little is known about the target areas and brain circuits of chronic ketamine administration.Anaesthetic dose and acute ketamine administration in adult lead to impaired working memory and executive function. Disorders of face emotion processing, working memory, language fluency and learning were observed. There is little report about chronic low dose of ketamine administration, especially the effects on brain function and physiological mechanism in adolescent. This study was designed to determine the consequences of chronic ketamine administration in adolescent cynomolgus monkeys following an exposure pattern that mimics adolescence drug abuse. The chronic drug effects on monkey's weight, behavior, brain functions and brain metabolism were investigated and the molecule mechanisms were explored.1. Objectives1.1. To observe body weight and behavioral changes in adolescent cynomolgus monkey following chronic low dose of ketamine exposure.1.2. To investigate the effects of chronic low dose of ketamine on brain function in adolescent cynomolgus monkey.1.3. To investigate the brain metabolism in striatum and frontal lobe following chronic low dose of ketamine exposure in adolescent cynomolgus monkey.1.4. To assay apoptosis in lentiform nucleus (LN) in adolescent cynomolgus monkey following chronic low dose of ketamine exposure.1.5. To examine expression of tyrosine hydroxylase (TH) in the prefrontal cortex (PFC) following chronic low dose of ketamine administration in adolescent cynomolgus monkey.2. Materials and methods2.1. Animal model establishedTwelve adolescent male cynomolgus monkeys were randomly divided into2 groups:eight to be administered with ketamine and four with saline as controls. A ketamine dose of1mg/kg in an injection volume of1ml (in saline) freshly prepared on the day of injection was given daily intravenously via arm vein under mild physical constraints for6months. Control monkeys were given sterile saline (1ml) daily.2.2. Body weight measurementAnimal body weights were recorded monthly to monitor the monkeys'well-being throughout the whole experiment.2.3. Behavioral observation15-min video recordings were made for each monkey after ketamine or saline injection at day1, day3, day7, day14, day56, day112, day183, day184and day185. Behavior observation including moving, walking, climbing and jumping was conducted.2.4. fMRIfMRI study was performed on186day. Respiration rate and body temperature were continuously monitored during the fMRI scan. For stimulation, the monkeys' right lower limb was moved (raised leg to60°) once per second. Average scanning time of fMRI for one monkey was about12min48s. Images were acquired in axial level in order to localization accurately.2.5. MRSMRS use single voxel PROBE PRESS point resolved spectroscopy. We examined15x15x10mm area in striatum and frontal lobe regions, avoiding skull and cerebrospinal fluid. Average scanning time of MRS for one monkey was about3min, and3times were performed.2.6. Apoptosis assayAfter fMRI scanning,1control and3ketamine-abuse monkeys were randomly chosen to be sacrificed. Brains were dissected and embedded in paraffin wax. Sections of left PFC and LN were cut coronally from paraffin blocks at4-um 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.2.7. Tyrosine hydroxylase (TH) For Immunohistochemistry TH positive cells in PFC was used to study morphological changes under light microscope. Brain tissues sections were treated with inactivate endogenous peroxidases. They were then blocked with1%bovine serum albuminand10%normal rabbit serum for1h, and then were incubated with horse radish peroxidase (HRP)-conjugated rabbit anti-goat IgG for1h. Finally, the sections were developed with a DAB kit. TH positive cells were assayed.3. Results3.1. Chronic low dose of ketamine administration inhibited the body weight increase in adolescent cynomolgus monkey following chronic ketamine exposure.There was no statistically significant difference between control and ketamine groups. But body weights of ketamine group increased more slowly compared with control group. Body weights increased for about10%in the control monkeys during the first2months, while the ketamine abuse monkeys took4months to gain the same10%increase in body weights. The mean difference at the end of6month was10%between the ketamine and control groups.3.2. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey depressed motor behaviorsFor behavior test, activities of moving, walking, climbing and jumping of ketamine group exhibited obvious decreased tendency as time went by. There were significant reduced activities of moving, walking and climbing of ketamine group in day183,184and185compared to control group (p<0.05).3.3. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey caused disturbance in the central nervous systemFirst, significantly higher levels of neural activities were observed in the right hemisphere of LN, fusiform gyrus (FG) and entorhinal cortex (Ent) in ketamine-challenged monkeys compared with controls. Second, monkeys in the control group had significantly higher neural activities in the substantia nigra (SN), ventral tegmental area (VTA), posterior cingulate cortex (CGp), visual cortex, and somatosensory cortex (SC) compared with ketamine-challenged monkeys.3.4. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey caused disturbance in brain metabolismIn MRS, Cho in striatum and frontal lobe in ketamine group are lower than those of control (p<0.05). Chronic ketamine abuse might affect cell metabolism such as membrane breakdown, turn over, synthesis, myelination and lipid metabolism in striatum and frontal lobe.3.5. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey did not cause obvious apoptosisThere were not significant differences of apoptotic cells between two groups in lentiform nucleus.3.6. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey decreased expression of TH protein in the prefrontal cortexTyrosine hydroxylase (TH) pilot experiment showed there was a qualitative reduction in the positively stained projective axon of dopaminergic neuron in the prefrontal cortex of monkeys with chronic ketamine abuse (p<0.01).4. Conclusion4.1. Chronic low dose of ketamine abuse inhibits the body weight increase in adolescent cynomolgus monkey following chronic ketamine exposure.4.2. Chronic low dose of ketamine abuse decreases locomotor activity, which suggesting induced tolerance after chronic ketamine administration in adolescent cynomolgus monkey.4.3. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey leads to depression in VTA and SN, which is correlated no drug seeking and behavior addiction. Significant brain activation in LN implicates a dysfunction of sensorimotor gating, which plausibly leading to motor inhibition. Activation in FG causes visual hallucination, which is regarded as the main reason of drug addicts. Activation in Ent-striatum may play an important role in schizophrenia and substance abuse. We identify the damage of SC firstly, which implicates injury function of somatosensory. This compensates the central action of ketamine, providing experiment proof and abstinence of ketamine abuse in adolescence.4.4. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey might affect neuroglial cell metabolism such as membrane turn over, synthesis and lipid metabolism in striatum and frontal lobe.4.5. There is no significant apoptosis. Perhaps chronic low dose of ketamine abuse in adolescent cynomolgus monkey induces other paths of apoptosis. Although apoptosis will take place at last, the procee of apoptosis was delayed.4.6. Chronic low dose of ketamine abuse impaires the function of prefrontal dopaminergic system, causing damage of cognitive function and decreased locomotor activity.