Long term ketamine and ketamine plus alcohol toxicity - what can we learn from animal models?

This review addressed the adverse effects of the frequently-used recreational drug, ketamine through using mice and monkey models. Our laboratory has documented initially that ketamine can induce the formation of hyperphosphorlated tau (hypertau), which is a hallmark of Alzheimer's disease (AD), in the cerebral cortex of both mice and monkeys as well as apoptosis in neurons in these species. Besides the cerebral cortex, other centers in the central nervous system (CNS) and peripheral nervous system (PNS) are also influenced by ketamine. Cerebellum was found to be down-regulated in both mice and humans after long-term of ketamine administration and it was caused by the apoptosis of Purkinje cells. Deleterious effects in other organs reported in long-term ketamine users include of kidney dysfunction leading to proteinuria, fibrosis of the urinary bladder and reduction in size of the urinary bladder leading to frequent urination, increase of liver fibrosis and cardiac problems such as premature ventricular beats. Moreover, ketamine is usually co-administrated with other chemicals such as caffeine or alcohol. It has been reported increased harmful effects when ketamine was used in combination with the above substances. Mechanisms of damages of ketamine might be due to 1) up-regulation of NMDA receptors leading to overestimation of glutamatergic system or 2) the metabolite of ketamine which was a hydroquinone exerted toxicity.

Extract of white button mushroom affects skin healing and angiogenesis.

White button mushroom extract was examined in this study on (1) its potential effect on angiogenesis in chorioallantoic culture and (2) its recovering effect on the skin after injury in the ICR mice. Methods used included TUNEL assay on apoptosis, immunohistochemistry for vascular endothelial growth factor (VEGF), proliferative cell nuclear antigen (PCNA), epidermal growth factor (EGF), transforming growth factor ß (TGF-ß), and immune factor CD4 and western blotting. The results of chorioallantoic culture showed that the mushroom treatment led to significant increase in densities of VEGF sites. In the skin injury, ICR mice model increased EGF, PCNA, and collagen fibers, along with decrease of TUNEL positive apoptotic cells and limited reaction of TGF-ß and CD4 indicated that white button mushroom extract appeared to have beneficial effects on skin in regeneration and after injury.

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.

How would composite traditional Chinese medicine protect the brain--an example of the composite formula "Pien Tze Huang".

Chinese medicine has a long history of several thousand years. The main form of Traditional Chinese Medicine (TCM) is composite, i.e. a mixture of up to 10 medicinal products. Thus a composite prescription of 4-5 kinds of Chinese medicinal products may contain several hundred kinds of chemical composition. The active ingredients and clinical efficacy of which are difficult to characterize. We aim to review the Chinese literature of TCMs with neuroprotective effects. We illustrate with our study on Pien Tze Huang (PZH) the use of in vivo tests in the study of composite TCM. Our results show evidence that PZH might have neuropreventive effects in rats.

Permanent deficits in brain functions caused by long-term ketamine treatment in mice.

Ketamine, an injectable anesthetic, is also a popular recreational drug used by young adults worldwide. Ketamine is a non-competitive antagonist of N-methyl-d-aspartate receptor, which plays important roles in synaptic plasticity and neuronal learning. Most previous studies have examined the immediate and short-term effects of ketamine, which include learning and cognitive deficits plus impairment of working memory, whereas little is known about the long-term effects of repeated ketamine injections of common or usual recreational doses. Therefore, we aimed to evaluate the deficits in brain functions with behavioral tests, including wire hang, hot plate and water maze tests, plus examine prefrontal cortex apoptotic markers, including Bax, Bcl-2 and caspase-3, in mice treated with 6 months of daily ketamine administration. In our study, following 6 months of ketamine injection, mice showed significant deterioration in neuromuscular strength and nociception 4 hours post-dose, but learning and working memory were not affected nor was there significant apoptosis in the prefrontal cortex. Our research revealed the important clinical finding that long-term ketamine abuse with usual recreational doses can detrimentally affect neuromuscular strength and nociception as part of measurable, stable and persistent deficits in brain function.

Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine.

Ketamine, a non-competitive antagonist at the glutamatergic N-methyl-d-aspartate (NMDA) receptor, might impair memory function of the brain. Loss of memory is also a characteristic of aging and Alzheimer's disease. Hyperphosphorylation of tau is an early event in the aging process and Alzheimer's disease. Therefore, we aimed to find out whether long-term ketmaine administration is related to hyperphosphorylation of tau or not in the brains of mice and monkeys. Results showed that after 6 months' administration of ketamine, in the prefrontal and entorhinal cortical sections of mouse and monkey brains, there were significant increases of positive sites for the hyperphosphorylated tau protein as compared to the control animals receiving no ketamine administration. Furthermore, about 15% of hyperphosphorylated tau positive cells were also positively labeled by terminal dUTP nick end labeling (TUNEL) indicating there might be a relationship between hyperphosphorylation of tau and apoptosis. Therefore, the long-term ketamine toxicity might involve neurodegenerative process similar to that of aging and/or Alzheimer's disease.

Mice are prone to kidney pathology after prolonged ketamine addiction.

ICR mice were injected with ketamine for 1, 3 and 6 months and the kidneys and urinary bladders were excised and processed for histology. Starting from 1 month, all addicted mice showed invasion of mononuclear white cells, either surrounding the glomerulus or the other tubules in the kidney. The aggregation of these cells extended all the way to the pelvis and ureter. As well, in the urinary bladder, the epithelium became thin and there was submucosal infiltration of mononuclear inflammatory cells. Silver staining revealed a loss of nerve fibers amongst the muscles of the urinary bladder of the treated. Immunohistochemistry on choline acetyltransferase which is a marker for cholinergic neurons also demonstrated a decrease of those cells. We hypothesized that prolonged ketamine addiction resulted in the animals prone to urinary infection.

Cell death in the Purkinje cells of the cerebellum of senescence accelerated mouse (SAMP(8)).

The cerebella of SAMP(8) (accelerated aging mouse) and SAMR(1) controls were analyzed by Western Blotting of tyrosine hydroxylase and choline acetyltransferase, as well as by TUNEL and histological silver staining. Both tyrosine hydroxylase and choline acetyltransferase levels were higher in SAMR(1) than in SAMP(8). There was also an age-related decrease in enzyme levels in SAMP(8), with the reduction of tyrosine hydroxylase being more apparent. Concomitantly, there was an age-related increase of apoptosis in the medial neocerebellum and the vermis as revealed by TUNEL, with changes being significant in the SAMP(8) strain. Histologically, some Purkinje cells appeared to disappear during aging. Taken together, the data suggests that the aging SAMP(8) strain displays differential Purkinje cell death in the medial cerebellum and that some of the dying cells are likely to be catecholaminergic.

Vagus, hypoglossal, and median nerves in human development.

In spite of the wealth of literature on the changes of the neurons in development of the brainstem and the spinal cord in vertebrates, the alterations of the cranial nerves and somatic nerves during the prenatal period was largely neglected. Particularly in humans, little information was available. The article reports the changes in the vagus, hypoglossal, and median nerves in the fetus and term babies. The changes of proportion of different-sized nerve fibers are documented. The patterns were different in the three nerves and the hypoglossal nerve seemed to show "pruning" of fibers during this period.

Immunohistological evidences of Ginkgo biloba extract altering Bax to Bcl-2 expression ratio in the hippocampus and motor cortex of senescence accelerated mice.

Ginkgo biloba extract 761 appears to display neuroprotective effect in many nervous diseases and aging. Deterioration of mental functions during aging is always accompanied by loss of neurons, presumably through apoptosis. Here, we studied the effect of G. biloba extract in the expression of Bax/Bcl-2 ratio, an important apoptotic index, in the hippocampus and motor cortex of the aging brain. Bax and Bcl-2 expressions were examined with immunohistochemical methods. Senescence Accelerated Mice Prone Strain 8 was used because the aging process was accelerated with neuropathological alterations similar to those found in the aging human brain. The mice were fed with either G. biloba extract or sucrose from the age of 3 weeks until sacrifice at 3 or 9 months old. In the hippocampus of G. biloba fed 9-month-old mice, the ratio of Bax positive cell to Bcl-2 positive cell (Bax/Bcl-2 expression ratio) was 11.43 +/- 3.11 (mean +/- SD); significantly lower (P < 0.05) than the Bax/Bcl-2 expression ratio of 20.99 +/- 5.34 in the sucrose fed mice. The Bax/Bcl-2 expression cell ratios, however, in the motor cortex were not significantly different between the two groups (2.22 +/- 1.35 versus 2.27 +/- 2.02 for the G. biloba and the sucrose fed mice, respectively). The decrease in the Bax/Bcl-2 expression cell ratio following G. biloba treatment might hence be able to protect the aging hippocampus from moving further down the apoptotic pathway. Western blotting confirmed the decrease of Bax in the brain even after a short term and high dose Ginkgo treatment. It is speculated that the G. biloba extract may be a potential neuroprotective agent against apoptosis through the differential expressions of the Bax and Bcl-2 in the hippocampus.

Effect of methotrexate on the intracellular phosphoribosyl pyrophosphate level and glucose transport of Ehrlich ascites tumor cells in vitro.

Methotrexate (MTX) suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. The intracellular level of phosphoribosyl 5-pyrophosphate (PRPP) of EAT cells increased in a dose-dependent manner in response to MTX treatment. At the same time, the rate of glucose transport was lowered. Hypoxanthine reversed both these effects of MTX and partially rescued EAT cell growth. Under all conditions tested, changes in rate of glucose transport were shown to be the result of alterations in the number of glucose transporter (Vmax) rather than ligand affinity (Km). The dual action of MTX as a chemotherapeutic agent is discussed in this light.

Human tumour necrosis factor-alpha inhibits glucose transport in cultured Ehrlich ascites tumour cells.

Human recombinant tumour necrosis factor-alpha (rhTNF-alpha) arrested the growth of Ehrlich ascites tumour (EAT) cells in vitro. It suppressed cellular glucose uptake and decreased the membrane density of glucose transporters as measured by glucose-reversible cytochalasin B binding. The glucose transporters' affinity for substrate was also reduced. However, rhTNF-alpha treatment exerted no effect on the phosphoribosyl pyrophosphate level in EAT cells. The role of rhTNF-alpha on the inhibition of glucose transport of tumour cells is discussed.

Analysis of Epstein-Barr virus in localized nasopharyngeal carcinoma tumors.

BACKGROUND: An f variant of Epstein-Barr virus (EBV) appears associated with nasopharyngeal carcinoma (NPC) in Southern Chinese. Early diagnosis of the tumor allows the detection of some localized tumors. METHODS: A polymerase chain reaction (PCR) assay for genotyping EBV was used to evaluate the presence of the virus in NPC biopsies of local tumors of eight Chinese patients. RESULTS: The f variant was detected in the nasopharynx of seven of eight patients. The f variant was present in equal frequency in the "normal" and tumor regions. CONCLUSIONS: Examination of localized NPC tumors by the PCR genotyping assay revealed EBV was present on the tumor side of the nasopharynx in greater quantities than the "normal" side in seven of eight patients studied. Concurrent infection with both the prototype F and f variant was observed in two of the eight patients investigated.

Direct detection of Epstein-Barr virus in peripheral blood and comparison of Epstein-Barr virus genotypes present in direct specimens and lymphoblastoid cell lines established from nasopharyngeal carcinoma patients and healthy carriers in Hong Kong.

By means of a PCR assay, EBV was demonstrated directly in peripheral blood of previously infected individuals. The virus was detected in approximately 80% of specimens from EBV-seropositive individuals, but not in cord-blood lymphocytes by this method. When virus present in peripheral blood was compared to that observed directly in NPC biopsies or throat washings, it was distinct from that seen in biopsies in 4/15 cases (27%) and from that seen in throat washes in 1/22 cases (5%). The throat-wash virus differed from the biopsy virus in 3/20 cases (15%). The prototype F virus was found in 7/10 LCLs (70%) established from NPC patients' peripheral blood, but was only detected in 2/9 specimens (22%) directly analyzed by the PCR assay. This finding suggests selective isolation of prototype F EBV in spontaneous LCLs established from NPC patients.

Detection and prevalence of the "f" variant of Epstein-Barr virus in southern China.

The "f" variant of Epstein-Barr virus (EBV) may have an association with the development and/or maintenance of nasopharyngeal carcinoma (NPC) among Southern Chinese. This variant is detected at a higher frequency among individuals with elevated IgA antibody levels against EBV capsid antigen who have no detectable NPC and in NPC patients as compared to healthy individuals or patients who are in remission for NPC for over 3 years. Conversion or replacement of the f variant by the prototype BamHI F virus usually occurs by 3 to 4 years after radiotherapy. By 5 years post-therapy the majority of people in remission for NPC no longer harbor the f variant in their oropharynx. Eradication of this f variant, however, does not appear essential for maintenance of a disease-free state since several patients harboring this variant were in remission for NPC for up to 21 years. The virus strain detected directly in the nasopharynx is not always identical to that seen in the oropharynx. Dual infection was commonly observed in throat washings of NPC patients although the biopsy from tumors harbored unique strains of EBV.

Direct identification of serotypes of natural human rotavirus isolates by hybridization using cDNA probes derived from segment 9 of the rotavirus genome.

Under stringent hybridization conditions, cDNA of segment 9 of the rotavirus genome, which codes for the viral protein VP7, permitted differentiation of serotypes of culture-grown rotaviruses and natural isolates of human rotaviruses directly from clinical specimens. This was evident for the following reasons. (i) The cDNA of one serotype selectively hybridized with the RNA of the same serotype of culture-grown rotaviruses. (ii) Natural isolates of the virus thus identified as serotype 2 were also those which gave the short electrophoretic pattern characteristic of the genome of serotype 2, subgroup 1 viruses. Isolates having the long electrophoretic pattern characteristic of the genome of subgroup 2 viruses were identified as serotype 1, 3, or 4 by this method. (iii) For patients who had previously undergone serological analysis, the serotypes being excreted were the same serotypes against which there was the most marked serum neutralizing antibody response. Although the virus population was genetically diverse, the preponderance of the population prevalent in Guangzhou and Foshan in each of the three successive years between 1982 and 1985 comprised a single serotype. The dominant serotype changed from one year to another, but there was minimal cocirculation of different serotypes in this community. All virus isolates belonged to serotype 1,2,3, or 4, and there was no evidence to suggest that the other human rotavirus serotypes were prevalent in this community.

In situ detection of Epstein-Barr virus markers in nasopharyngeal carcinoma patients.

Cryosections of nasopharyngeal tissue from 9 patients suspected of having nasopharyngeal carcinoma (NPC) were examined for the presence of Epstein-Barr virus (EBV) markers in situ to assess virus infection in the nasopharynx. Viral DNA, EB nuclear antigen, and/or early antigens (EA.D and EA.R) were detected in 5 NPC specimens. EBV infection was not confined to the tumor areas of the biopsy specimens. Lymphoid cells and nontumor areas of these specimens contained EBV markers. In addition, nasopharyngeal tissues obtained from 3 of 4 patients in clinical remission for the disease showed evidence of EBV infection.

Effect of hyperthermia on growth of Ehrlich ascites tumor cells.

Hyperthermic treatment at 43 degrees C suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. Incubation of EAT cells at 43 degrees C for as little as 1.5 h totally abolished the transplantability of the tumor. At the same time, the rate of cellular glucose uptake, the density of glucose transporter on the cells as well as the extent of thymidine, uridine and leucine incorporation were significantly reduced.

Interrelationship of number of glucose carriers and intracellular phosphoribosyl diphosphate level of Ehrlich ascites tumor cells in vitro.

The intracellular phosphoribosyl diphosphate (prpp) levels of Ehrlich ascites tumor cells increased during glucose supplementation and decreased during glucose deprivation, while the numbers of glucose carriers as determined by glucose-reversible cytochalasin-B binding changed in an opposite manner relating to the extracellular glucose concentrations and the intracellular prpp levels of Ehrlich ascites tumor cells in vitro. Incubation of cells with hypoxanthine or 2,4-dinitrophenol lowered the intracellular prpp levels and resulted in an increase in numbers of glucose carriers.

Intracellular phosphoribosyl diphosphate level and glucose carrier in Ehrlich ascites tumour cells during tumour growth and diabetes in tumour-bearing mice.

The ability of Ehrlich ascites tumour (EAT) cells in mice to take up glucose as well as the density of glucose carriers on the cells increased progressively during the course of tumour development. Simultaneously as the rate of uptake rose, the intracellular phosphoribosyl diphosphate (PRPP) levels dropped responding to the decrease of serum glucose. Hyperglycaemia induced in the host by alloxan or streptozotocin administration increased the serum glucose concentrations and intracellular PRPP levels but decreased the density of glucose carriers of the cells, whereas insulin administration reversed this condition. The physiological significance of these observations are discussed.