Maria S.M. Wai

Chronic Ketamine Administration Modulates Midbrain Dopamine System in Mice

Ketamine is an anesthetic and a popular abusive drug. As an anesthetic, effects of ketamine on glutamate and GABA transmission have been well documented but little is known about its long-term effects on the dopamine system. In the present study, the effects of ketamine on dopamine were studied in vitro and in vivo. In pheochromocytoma (PC 12) cells and NGF differentiated-PC 12 cells, ketamine decreased the cell viability while increasing dopamine (DA) concentrations in a dose-related manner. However, ketamine did not affect the expression of genes involved in DA synthesis. In the long-term (3 months) ketamine treated mice, significant increases of DA contents were found in the midbrain. Increased DA concentrations were further supported by up-regulation of tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Activation of midbrain dopaminergic neurons could be related to ketamine modulated cortical-subcortical glutamate connections. Using western blotting, significant increases in BDNF protein levels were found in the midbrain, suggesting that perhaps BDNF pathways in the cortical-subcortical connections might contribute to the long-term ketamine induced TH upregulation. These data suggest that long-term ketamine abuse caused a delayed and persistent upregulation of subcortical DA systems, which may contribute to the altered mental status in ketamine abusers.

Ketamine effects on the urogenital system--changes in the urinary bladder and sperm motility.

Different doses of ketamine (10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, and 60 mg/kg) were injected i.p. (I.P.), respectively, to male ICR mice to determine the optimal dosage for chronic administration. At and above 40 mg/kg I.P. injection, mice had almost no hindlimb movement during swimming test. Subsequently, 30 mg/kg was used as the dose for the study in the toxicity of long‐term ketamine administration on urinary bladder and sperm motility. The treatment group were subdivided into two (n = 10 each group); one received daily ketamine treatment i.p. for 3 months and another group for 6 months. Corresponding number of mice in control groups (n = 5 each group) received saline injection instead of ketamine. Terminal dUTP nick and labeling (TUNEL) study and Sirius red staining were carried out on the sectioned slides of the urinary bladders to study the degree of apoptosis in both epithelium and muscular layers of the urinary bladder and the relative thickness of the muscular layers in this organ was also computed. Apoptosis in the bladder epithelium was observed initially in the 3‐month ketamine treated mice and the number of apoptotic cells was significantly different (P < 0.05) between the 3‐month and 6‐month ketamine treated mice and the control. The relative thickness of muscular layers in the bladder wall also decreased significantly (P < 0.05) when the 6‐month treated mice and the control were compared. Sirius red staining revealed increase of collagen in the urinary bladder of the treated mice, most evidently 6 months after ketamine treatment. In addition, the sperm motility was studied and there was a statistically significant difference between the control and ketamine treated groups in the percentages of sperms which were motile (P < 0.05). This suggested that the chronic administration of ketamine affected the genital system as well. Microsc. Res. Tech., 2011.

Exogenous progesterone: a potential therapeutic candidate in CNS injury and neurodegeneration.

The role of progesterone (PROG) in the regulation of reproductive behavior is well understood, but a large and growing body of evidence indicates that this hormone also exerts neuroprotective effects on the central nervous system (CNS), i.e. in spinal cord injuries, traumatic brain injuries and in the age-related pathological process. Its neuroprotective actions, now well documented by experimental studies, make it a particularly promising therapeutic agent for neuroinjury and neurodegenerative diseases. The purpose of this article is to review recent preclinical and epidemiological evidences that exogenous administration of PROG or its metabolites plays an important role in the CNS. The diverse signaling mechanisms and the dose- dependent neuroprotective actions of PROG are also summarized. Awareness of the pleiotropic effects of PROG may open a novel perspective for the treatment of injuries and diseases in the nervous system. PROG could be produced in the brain by neurons and glial cells in the CNS of both male and female. Laboratories around the world have reported that administering relatively large doses of PROG during the first few hours or even days after injury significantly limits CNS damage, reduces loss of neuronal tissue and improves functional recovery. PROG appears to exert its protective effects by protecting or rebuilding the blood-brain barrier, decreasing the development of cerebral edema, down-regulating the inflammatory cascade, and limiting cellular necrosis and apoptosis. All these are plausible mechanisms of neuroprotection.

Effects of DL-3-n-Butylphthalide on Vascular Dementia and Angiogenesis

Abstract3-n-Butylphthalide (NBP) is a compound extracted from Chinese celery and is used as an anti-hypertensive herbal medicine for treating stroke patients. The aim of this study is to demonstrate the effects and mechanisms of this compound through in vitro and in vivo experiments. Culture experiments were performed by adding hydrogen peroxide (H2O2) to SH-SY5Y cells. From the MTT assay result, enhanced cell survival was observed with DL-NBP treatment, regardless of whether they are added before, simultaneously with or after the addition of H2O2. For the in vivo experiment, Spontaneously Hypertensive rats and Wistar Kyoto control rats with chronic cerebral ischemia, which were induced by bilateral transection of the common carotid arteries, were given DL-NBP. Their performances in the place navigation test and spatial probe test in the Morris Water Maze have significantly improved compared with the DL-NBP untreated animals, indicating an improvement in spatial learning and memory in the ischemic-animals. In addition, in the chick embryonic chorioallantoic membrane assay, angiogenesis was more vigorous under the effects of DL-NBP, together with increased expression of growth factors, VEGF, VEGF-receptor and bFGF. All these suggested that one of the mechanisms of DL-NBP might be ameliorating vascular dementia and promoting angiogenesis.

DL-3-n-Butylphthalide, an anti-oxidant agent, prevents neurological deficits and cerebral injury following stroke per functional analysis, magnetic resonance imaging and histological assessment.

DL-3-n-Butylphthalide (NBP) is a synthetic compound based on L-3-n-Butylphthalide which was isolated from seeds of Apium graveolens. The present study aims at evaluating the outcome of NBP given prior to and after the onset of ischemic stroke in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Stroke was induced by the middle cerebral artery occlusion (MCAO) in SHR and WKY. For pre-treatment, NBP was administered to SHR and WKY daily for two months prior to MCAO. For post-treatment, NBP was given daily for seven consecutive days after MCAO. Seven days post-surgery, rats were tested for the presence of neurological deficits. Magnetic resonance imaging (MRI) and 2,3,5-triphenyltetrazolium chloride (TTC) staining were employed to calculate the infarct volume. The cerebral cortex and corpus striatum in the ischemic penumbra area were examined microscopically for pathological changes. In SHR, NBP pre- and post-treatment significantly lowered neurological deficit scores, reduced infarct volume, and minimized pathological changes in the penumbra area when compared to oil-vehicle treated controls. In WKY, these beneficial effects were observed only in the post-treatment group. The beneficial effects of NBP post-treatment were greater in WKY than in SHR. Results indicated that NBP could exert both preventive and therapeutic effects on ischemic stroke in SHR, but only exerted therapeutic effect in WKY.

Changes of apoptosis-related proteins in hippocampus of SAM mouse in development and aging

Expression of Caspase and Bcl-2 proteins was examined in the hippocampus of senescence-accelerated mice (SAM, P8 and R1 strain) from E19 to 16 months of age. Immunoblotting analysis showed no upregulation of pro-apoptotic proteins (caspase-2L, -3, -6, -8, -9, and Bax) with age while all the anti-apoptotic proteins (caspase-2S, Bcl-2, and Bcl-XL) remained unchanged during aging. Terminal dUTP nick end labeling (TUNEL) and electron microscopy on the hippocampus of 3- and 16-month-old SAM revealed very few TUNEL positive cells in both groups. Morphometric study further showed neuronal loss in the hippocampus was not age-related. Our results suggest apoptosis and cell loss are minor events in the hippocampus of SAM mice and are unlikely to be the cause of functional decline during aging in SAM.

Downregulation in the human and mice cerebella after ketamine versus ketamine plus ethanol treatment

To study the deleterious effects of ketamine and the potential interaction effects between ethanol and ketamine on the cerebellum, functional magnetic resonance imaging (fMRI) tests were performed on the habitual ketamine users (n = 3) when they flexed and extended their upper limbs. Another fMRI test was performed on the same participants in which they consumed alcohol (12%, 200 mL) 1 h before the test. Downregulation on the activity of cerebellum was observed and the level of activation in the cerebellum decreased dramatically in habitual ketamine users with alcohol consumption before the test. Further studies were performed by using male ICR mice receiving treatment of ketamine only [30 mg kg−1 intraperitoneally (i.p.)] or ethanol only everyday (0.5 mL 12% orally) and those with coadministration of the above dosages of ketamine and ethanol for 3 months. Fewer Purkinje cells were observed in the cerebellar sections of ketamine treated mice under silver staining. For TUNEL test, a significant increase in the apoptotic cells were observed in the cerebella of the ketamine treated mice (P = 0.016) and of those with co‐administration of ketamine and ethanol (P < 0.001), when compared with the control. A statistical significance (P < 0.001) in two‐way ANOVA test indicated that there might be an interactive mechanism between ethanol and ketamine acting on the cerebellum. Microsc. Res. Tech. 2011.

Cardiotoxicity induced in mice by long term ketamine and ketamine plus alcohol treatment

Increased use of ketamine as a recreational drug in rave party has raised lots of social concerns. Although its toxicity on the central nervous system (CNS) and urinary system had been reported, its potential adverse effects on the heart is still not addressed. Using our long term ketamine addiction model in which, 1-month-old ICR mice were given ketamine intraperitoneally (i.p. 30 mg/kg) for different study periods (6 weeks, 16 weeks and 28 weeks). The electrocardiogram (ECG) and the level of troponin as well as the presence of lactate dehydrogenase were used together to determine the cardiac function of mice. ST elevations were observed in 30% of mice from 6 weeks onwards. Concomitant increase in troponin in serum and presence of lactate dehydrogenase positive cardiac cells were taken together to indicate cardiac damages. The potential interactive effects of alcohol (ethanol) and ketamine were also addressed and 0.5 ml of 10% ethanol was administrated additionally to the mice daily and orally for the last 4 weeks in the 28-week long-term ketamine treated animals. The cardiac functions of these mice deteriorated more severely as indicated by further rise in troponin levels. In addition, high number of lactate dehydrogenase positive cardiac cells and abnormal ECGs in both ketamine and ketamine plus alcohol treated models also gave coherent conclusions.

The pattern of c-fos activation in the CNS is related to behavior in the mudskipper, Periophthalmus cantonensis

The effects of three types of behavior on c-fos activation in different brain regions of the mudskipper, Periophthalmus cantonensis, were studied by immunocytochemistry. Animals were divided into four groups: The control group did not undergo any specific treatment. The second group consisted of animals that were agitated for an hour with a glass rod at an irregular speed. The third group contained animals showing aggressive behavior during an hour of monitoring, i.e. documented territorial hostility by raising of dorsal fins and pursuit of intruders. In contrast to these three groups, which had last been fed 24h earlier, the fourth group included animals that had been nourished 1h prior to sacrifice. Results showed that, in most brain regions of control animals, there were relatively few c-fos positive cells. After fish had been agitated, however, very prominent c-fos label was seen in the lateral and medial parts of the telencephalon, the thalamus, hypothalamus, pituitary and medulla. In aggressive fish, a significant increase in the number of c-fos positive sites, as compared to control fish, was observed in the diencephalons, pons and medulla, but not in the telencephalon. After feeding, there was a less substantial increase in c-fos protein expression in the diencephalon, but an even more prominent c-fos activation in the pons and medulla. Our present results support the hypothesis that, in fish, the medial telencephalon is involved in avoidance reaction and the lateral telencephalon in spatial memory, whereas rhombencephalic activation may reflect activity of cranial nerve nuclei.

Profiles of serotonin receptors in the developing human thalamus

The critical importance of the thalamus and its serotonergic innervation with respect to neuropsychiatric syndromes is increasingly recognized. This study investigates the localization of serotonin (5-hydroxytryptamine; 5-HT) receptors by immunohistochemistry in the thalamic nuclei of human fetuses aged 21 to 32 weeks of gestation. Results indicate that, already at 21 weeks of gestation, two 5-HT receptors are present in the dorsomedial nucleus of the developing thalamus: 5-HT2A receptors are localized in neurons and 5-HT2C receptors in fibers. By 31 and 32 weeks of gestation, 5-HT1A and 5-HT4 receptors are also detected in neuronal fibers of the same nucleus. At this later developmental stage, the percentage of 5-HT2A labeled neurons has significantly increased in the dorsomedial nucleus, and 5-HT2C positive neurons are observed in the centromedian and lateroventral thalamic nuclei as well. In contrast, neither neuronal cells nor fibers display any immunoreactivity for 5-HT3 or 5-HT6 receptors at any of the ages examined. Our observation that 5-HT1A, 5-HT2A, 5-HT2C and 5-HT4 receptors are present in the human thalamus prenatally indicates that 5-HT may play a role during fetal development. Disrupted development of the thalamic serotonergic system during this gestational period may contribute to the pathophysiology of neuropsychiatric disorders.