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Abstract

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.

Keywords

noncompetitive NMDA antagonist prefrontal cortex behavioral test apoptosis brain deficits

Introduction

Ketamine was first synthesized in 1962 as a short-acting anesthetic in human and veterinary medicine.^1,2 It produces a unique anesthetic state called dissociative anesthesia. When ketamine is used in smaller doses, it can produce a psychedelic experience of incredible intensity.^2,3 Because of these side reactions, it has become increasingly popular in recent years as a recreational drug in night-clubs.^{4
–6} Recent surveys suggested that 10% of drug users in the United Kingdom use ketamine on a regular basis in night-clubs.⁷ The percentage of ketamine users has rapidly increased 4-fold between 1997 and 2001 among night-club patrons, particularly among young people. Currently, Ketamine is a controlled (Schedule III) substance in the United States (http://www.anestesia.com.mx/articulo/keta.html) and other countries (http://laws.justice.gc.ca/eng/C-38.8/20100120/index.html?rp2=HOME&rp3=SI&rp1=ketamine&rp4=all&rp9=cs&rp10=L&rp13=50).

Ketamine is a non-competitive antagonist of N-methyl-d-aspartate (NMDA) receptor, which is considered to be one of the vitally important receptors in mechanisms of synaptic plasticity and neuronal learning, such as long-term potentiation and long-term depression.⁸ Age-related decrease in NMDA receptor function is also related to memory decline in the elderly.⁹ Ketamine-mediated hypofunction of NMDA-receptor results in a combination of enhancement of thalamo-limbic systems and inhibition of thalamo-neocortical systems.^1,10 NMDA receptor antagonist with repetitive ketamine administration has been shown to reduce the function of the prefrontal dopaminergic system in rats, which plays a critical role in sustaining working memory tasks and executive functions.¹¹ Most previous studies have examined the immediate and short-term effects of ketamine in healthy individuals, resulting in impairment of working memory, learning and cognition,¹² whereas little is known about the long-term effects of repeated ketamine administration on the brain. Recently, some studies performed with rodents have shown that prolonged exposure to ketamine increased cell death in brain areas including the prefrontal cortex of the central nervous system (CNS).^11,13 These findings suggested that chronic exposure to ketamine in the CNS might interfere with learning and memory through a neurotoxic effect related to activation of apoptotic pathways. Prefrontal cortex is implicated in a variety of cognitive and executive processes, including working memory, decision-making, inhibitory response control, attentional set shifting and temporal integration of voluntary behavior constituting the so-called ‘pleasure centers.’^{14

–19} All of these abilities depend on proper prefrontal cortex neuronal network connections, which are highly sensitive to their neurochemical environment. Disruption to this neurochemical environment would obviously damage the functions of prefrontal cortex, but the effect of long-term ketamine (as an NMDA receptor antagonist) on prefrontal cortex is still largely unknown.²⁰

In view of the importance of prefrontal cortex, our study explored the effects of long-term ketamine administration on functional changes in mice prefrontal cortex, including sensorimotor and cognitive performance using behavioral tests, as well as the changes in expressions of proteins regulating the extrinsic (caspase-3) and the mitochondrial (Bcl-2 and Bax) apoptotic pathways. We hypothesized that long-term ketamine use could permanently damage the sensorimotor processes of prefrontal cortex, therefore we investigated whether the damage was related to apoptosis or not in our study of the long-term effects of repeated ketamine injection of usual recreational dose on the mouse brain.

Materials and methods

Experimental animals, groups and treatments

Four-week old male ICR (Institute of Cancer Research) mice bred in the Chinese University of Hong Kong (CUHK) animal facility were used because rodents are extensively used in studies of ketamine effects^11,21 and the appropriate facilities were available for the behavioral tests. Mice were housed 10 per cage with water and food pellets available ad libitum in a room kept at 22 ± 1°C and exposed to 12:12 hour (h) light/dark cycles. This study was approved by the Institutional Animal Care Committee of CUHK. For evaluation of the behavioral and neurochemical effects induced by chronic ketamine administration, 90 mice were randomly divided into 3 groups receiving treatment doses of 1-, 3- or 6-month of daily intra-peritoneal injections of ketamine at 30 mg/kg (Alfasan ketamine 10% injectables, Holland) or placebo of equal amounts of saline. We used a sub-anesthetic dose of 30 mg/kg of ketamine, which is regarded as the recreational dose for rodents with a LD₅₀ of 600 mg/kg⁴ and is consistent with dosages reported in the literature.^21,22

In each group of 30 mice, 20 were injected with ketamine and 10 with saline as controls. The weights of mice were recorded every week for adjustment of ketamine injection and monitoring of the well-being of the animals. At the end of 1, 3 or 6 months, all mice were used for the behavioral tests and then sacrificed for subsequent analysis of brain samples. In each of the 1-, 3- or 6-month groups, 3 control and 6 ketamine-treated mice were used for terminal dUTP nick end labeling (TUNEL) staining, and 7 control and 14 ketamine-treated mice were used for prefrontal cortex samples for Western blot assay.

Behavioral studies

We performed the following three behavioral tests 4 h after the administration of ketamine or saline on the final day of each treatment or control group, with the water maze test requiring training and testing on consecutive 3 days immediately before the final day. Because of the short half-life, ketamine and its metabolites are cleared by urinary excretion 4 h after the dose.^1,2 The aim was to study effects due to possible stable and persistent damages to the brain caused by ketamine rather than its immediate effect after ketamine injection.

Wire hang test

Neuromuscular strength was evaluated by placing the animals on a wire cage lid.^23,24 The lid was then turned upside down approximately 50 centimeter (cm) above the surface of a soft bedding material and regularly waved (4 strokes per second (sec)) in the air so that the mouse had to grip the wire to avoid falling off. The latency time in sec to fall from the lid onto the bedding was recorded, with 60 sec as the cut-off time.

Hot plate test

Sensitivity to a painful stimulus was evaluated by the hot plate test, one of the most commonly used tests for determining the analgesic effects of experimental drugs in rodents.²⁵ The mouse was placed on the flat aluminum surface of the hot plate maintained at 50 ± 1°C. Ambulation of the mouse on the plate was limited by a 16 cm high non-opaque Plexiglas frame of 11 × 9 cm² area. The frame enclosed the surface so that the mouse could not jump out. The numbers of times the mouse licked its fore and hind paws, fluttered, shook or jumped up within 25 sec were recorded. We have observed that these are the four types of movements the mice would do in response to placement on a hot plate. After the trial, the animal was removed from the hot plate immediately to prevent tissue damage.

Water maze test

Swim tasks are used to measure spatial navigation learning and memory in mice.^{26
–28} A black circular tank of 150 cm diameter was filled to a depth of 25 cm of water and maintained at 23 ± 1°C. The water was changed for each day’s experiment. The animals were to learn the best swimming strategy to find the visible white platform exposed about 1 cm above the surface of the water in the center of the circular tank. The position of the platform remained the same throughout the whole experiment. Several prominent fluorescent cues were placed around the tank at four fixed points, with one in each quadrant, including a T-shape (the starting point where the mouse was put into the tank), a Y-shape, a moon and a triangle. During the experiment, these cues were intended to facilitate mouse spatial orientation in the dark environment when the tank was closed with a long curtain hung over it from the ceiling above. Each trial began with the mouse being placed at the starting point along the edge of the pool facing the wall to avoid seeing the platform. The escape latency time for the mouse to locate and climb onto the platform was recorded. If a mouse did not find the platform after 90 sec, it would be gently guided to the platform and allowed to stay on the platform for 10 sec to recognize the location. The mouse would have three such consecutive trials each day with 30 sec of rest time between trials. Escape latency time to reach the visible platform or 90 sec if failed was recorded. All groups of mice were trained for 3 days with three trials per day, starting 4 h after ketamine or saline injection, and were then tested on the day after the training, which was the final day of treatment/control in each group. We used this water maze test to test the working memory and visual acuity.^29,30

TUNEL assay

The DNA fragmentation indicative of apoptosis was examined using TUNEL^31,32 that could detect early-stage apoptosis and examine the topographic distribution of apoptotic cells. Mice were sacrificed for TUNEL as we previously reported.³³ Mice were anesthetized with 0.5 mL of 7.0% chloral hydrate (1 mg/kg), and then exsanguinations were performed with 50 mL of saline through the cardiac apex of the left ventricle till the liver turned pale pink. Perfusion was continued with 50 mL of 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS; pH 7.4) through the same position. The PFA solution could keep the tissue’s shape from changing before embedding; then, various tissue samples were removed and immersed into 4% PFA for 24 h for further fixation. The tissue samples were then dehydrated and embedded in paraffin wax. Blocks were cut into 5 µm sections with a microtome.

TUNEL was performed using ApopTag^® Peroxidase In Situ Apoptosis Detection Kit (Millipore Corporation, Billerica, Massachusetts, USA) according to the manufacturer’s instruction including both positive and negative controls as we previously reported.³³ Sections were first deparaffinized, rehydrated and treated with proteinase K (20 μg/mL). After quenching with 3.0% hydrogen peroxide, the sections were treated with biotin-deoxyuridine triphosphate in the working solution of deoxynucleotidyl transferase (TdT) for 1 h at 37°C. The reaction was stopped with the stop/wash buffer. To detect the binding of dioxigenin-11-dUTP, sections were treated with the anti-digoxigenin conjugate for 30 min. Finally, after visualization with dimethylaminoazobenzene, apoptotic cells including the cellular DNA fragmentation were photographed with a digital camera (Zeiss Axiocam MRc5). In each assay, negative controls were included using the same incubation procedure but omitting the TdT, while positive controls were prepared by incubating the pretreated sections with DNase I (Sigma, DN 25, 1.0 μg/mL) that hydrolyzed DNA preferentially at sites adjacent to pyrimidine nucleotides. We counted TUNEL-positive cells from 10 sections of each mouse prefrontal cortex sample. Sections were selected from comparable layers of prefrontal cortex. A total area of at least 15 mm² was counted for each sample. Results were expressed in number of TUNEL positive cells per mm².

Western blot assay

Western blot assay was modified as we previously published.³⁴ For Western blot assay, mice were sacrificed as we previously reported.³⁵ Briefly, mice were killed by cervical dislocation. The prefrontal cortex was immediately removed and snap-frozen in liquid nitrogen and stored at −80°C until processed for Western blot assay. Brain tissue samples were homogenized in 0.5 mL of lysis buffer, 50 mM Tris–HCl (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, 0.1% sodium dodecyl sulphate (SDS), 1% Triton X-100, 0.1 mg/mL phenylmethylsulphonyl fluoride, 1 mg/mL leupeptin, 1 mg/mL pepstatin A and 5 mg/mL aprotinin. The homogenate was centrifuged at 14,000 g for 30 min at 4°C and the supernatant obtained was either immediately used or stored at −80°C until use. The protein concentration of the extract was determined by the Bio-Rad DC protein assay (500-0111, Bio-Rad Laboratories, Hercules, California).

Equal amounts of protein (100 µg) samples were boiled in 2 × Laemmli loading buffer (final concentrations: 0.0625 M Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.001% bromophenol blue)³⁶ for 6 min before loading on 12% SDS-polyacrylamide gel. After electrophoresis and semi-dry transfer, the nitrocellulose membrane was blocked (5% non-fat dry milk, 0.05% Tween-20 in PBS) and then incubated overnight at 4°C with either of the following anti-sera (dilution): actin (1:20,000) (MAB1501, Millipore Corp.), Bax (1:1,000; sc-526, Santa Cruz Biotechnology Inc., Santa Cruz, California), or Bcl-2 (1:1,000; sc-783, Santa Cruz Biotechnology Inc.), caspase-3 (1:1,000) (9662, Cell Signaling Technology Inc., Danvers, Massachusetts). The following day, after three sequential 5 min washes with 0.05% Tween-20 in PBS (PBST), the membrane was incubated with the appropriate IRDYE® FC conjugated secondary antibody 1:10000 for 1 h (IRDYE® 700DX anti-rabbit FC 605-430-003 for Bax, Bcl-2 and caspase-3; IRDYE® 800CW anti-mouse FC 611-131-003 for actin; LI-COR Biosciences, Lincoln, Nebraska 68504-0425). After washing three times for 10 min with PBST, the bound antibodies were then visualized and recorded using the ODYSSEY Infrared Imaging System (LI-COR Biosciences). Band density value of individual proteins was normalized to that of the actin of the same sample. Bax to Bcl-2 ration (Bax/Bcl-2) was calculated from the Bax and Bcl-2 results of the same sample.

Statistical method

All data were expressed as mean ± standard deviation (SD) or median (10th to 90th percentile) for the groups. One-month, 3-month and 6-month ketamine groups were compared to their respective control groups unless specified. The Wilcoxon or Student-t (two-tailed) were used when appropriate for non-normally or normally distributed datasets, respectively. Specifically, Student-t test was used for comparison of body weight and Western blot results between groups, while Wilcoxon test was used for comparisons of behavioral test and TUNEL results. The difference between groups was considered statistically significant when p-value was less than 0.05.

Results

Body weight of mice

Setting body weights at 0-month as the baseline of 100%, the percentage increases in body weight for both the ketamine and control groups at 1, 3 and 6 months were significant (p < 0.05; Figure 1). Although the percentage increases were less among all ketamine groups than the percentage increases of the respective control groups, for example, the percentage increase was 109.6% ± 9.9% (mean ± SD) in the 6-month ketamine group versus the 114.3% ± 8.9% in the 6-month control group, the differences in percentage increases between ketamine groups and their respective control groups were not statistically significant.

Figure 1.

Body weights of different mouse groups. The body weights of mice were recorded weekly. The final body weights were expressed as the percentage of the body weights at 0-month (taken as 100%) when the experiment started. All ketamine-treated groups showed less gains in body weights than the respective control groups; however, the differences were not statistically significant. Results were group means ± standard deviations.

Behavioral studies

All behavioral tests were performed 4 h after the last dose of ketamine, therefore, the results reflected not the immediate effects but the stable and persistent effects of long-term ketamine.

Wire hang test

The latency times (in sec) of falling from the wire for 1-, 3- and 6-months ketamine groups were consistently less than the latency times of their respective control groups (Figure 2). After 6 months' ketamine administration, the latency time of the 6-month ketamine group (6.0, 2.9−12.2 [median, 10th−90th percentile]) was significantly less (p < 0.05) than the 6-month control group (13.5, 4.8−20.9).

Figure 2.

Wire hang test results of different mouse groups. Wire hang test was performed 4 hours after the last ketamine or saline injection to the mouse. The 6-month ketamine group showed significantly shorter latency time (p < 0.05) to fall from the wire than the 6-month control group. Results were group medians + 90th percentiles.

Hot plate test

For the 6-month ketamine group, the total number of movements of the mice (14.0, 9.9−18.2 [median, 10th−90th percentile]) on the hotplate was significantly less (p < 0.05) than the number of the control group (19.5, 13.8−25.2; Figure 3). This showed that after 6 months of ketamine injection, the sensory perception of heat was damaged. However, there were no significant differences or consistent trends of change in ketamine-treated mice for 1 and 3 months as compared to their respective controls.

Figure 3.

Hot plate test results of different mouse groups. Hot plate test was performed 4 hours after the last ketamine or saline injection to the mouse. The 6-month ketamine group showed significantly less in the number of movements (p < 0.05) on the hot plate (50 ± 1°C) than the 6-month control group. Results were group medians + 90th percentiles.

Water maze test

The escape latency time (in sec) for the mice to locate and climb onto the platform showed no statistically significant differences between the 1-, 3- and 6-month ketamine groups as compared to their respective control groups (Figure 4), although the ketamine-treated mice consistently used more time to climb onto the platform in all groups.

Figure 4.

Water maze test results of different mouse groups. After training for 3 days, water maze test was performed 4 hours after the last ketamine or saline injection to the mouse. The escape latency time of all ketamine-treated groups were not significantly different from their respective control groups. Results were group medians + 90th percentiles.

TUNEL and Western blot assay

There were no statistically significant differences in the TUNEL-positive cell counts in the prefrontal cortex between the ketamine groups of 1-, 3- and 6-months and their respective control groups (Figure 5). Although Western blot results showed consistently that all 1-, 3- and 6-month ketamine groups had higher Bax, lower Bcl-2, higher Bax/Bcl-2 and higher caspase-3 (to actin ratio) levels in prefrontal cortex than their respective control groups (Table 1), these differences were not statistically significant. Representative images of Western blot results are shown in Figure 6 . The antibody for caspase-3 we used detects both the full length (35 kDa) and the cleaved (17 kDa) forms. The cleaved caspase-3, i.e. the active form, was not detected, indicating that apoptosis did not occur in the prefrontal cortex samples we took.

Figure 5.

Terminal dUTP nick end labeling (TUNEL) results of prefrontal cortex samples from different mouse groups. TUNEL-positive cell counts in the prefrontal cortex samples were determined. The cell counts were not significantly different between all ketamine-treated groups and their respective control groups. Results were group medians + 90th percentiles.

Table 1.

Western blot results of prefrontal cortex samples from different mouse groups. Bax, Bcl-2 and caspase-3 (to actin ratio) were performed and Bax/Bcl-2 was calculated from the Bax and Bcl-2 results of the same sample

Protein	1 Month		3 Months		6 Months
Protein	Control	Ketamine	Control	Ketamine	Control	Ketamine
Bax	0.0027 ± 0.0010	0.0042 ± 0.0026	0.0035 ± 0.0027	0.0040 ± 0.0026	0.0026 ± 0.0011	0.0031 ± 0.0018
Bcl-2	0.040 ± 0.025	0.024 ± 0.018	0.056 ± 0.031	0.032 ± 0.026	0.043 ± 0.029	0.030 ± 0.025
Caspase-3	0.053 ± 0.026	0.075 ± 0.036	0.040 ± 0.021	0.046 ± 0.018	0.031 ± 0.012	0.045 ± 0.022

^a Please note the consistent changes of higher Bax, lower Bcl-2, higher Bax/Bcl-2 and higher caspase-3 in ketamine-treated groups as compared with their respective control groups, suggesting a possible tendency of more apoptosis associated with ketamine. However, the differences were not statistically significant. Results were group means ± standard deviations (SD).

Figure 6.

Representative images of Western blot results for Bax, Bcl-2 and caspase-3 of prefrontal cortex samples from control and ketamine mice. Please note the cleaved caspase-3, i.e. the active form (17 kDa), was not detected. Only the full length caspase-3 (35 kDa) was visualized.

Discussion

Although there were no significant differences between the gains in body weights in ketamine groups as compared to the control groups, the consistently lower gain in body weight with ketamine treatment (Figure 1) may possibly indicate the presence of somatic effect associated with ketamine. The CNS plays important roles in the control of appetite and maintenance of body weight.^37,38 With long-term ketamine administration, disruption of brain function is not unexpected, with possible effects on the hormonal and neuronal signals for appetite and body weight control in young animals. Indeed, in another series of experiments (unpublished data) in our laboratory after 6 months ketamine administration to adolescent monkeys, functional magnetic resonance (FMR) imaging revealed that the ventral tegmental area (VTA) was damaged; VTA is involved in the reward circuitry for food intake.³⁷ This might occur in mice with long-term ketamine administration, affecting appetite and resulting in reduced body weight gain over time as compared to control groups. Further detailed studies are necessary to elucidate this possible somatic effect of ketamine using different dosages and examining the mechanism of action in more depth.

Although the somatic effect of ketamine was not truly significant in our study, the 6 months of treatment with ketamine showed statistically significant and permanent impairments in neuromuscular strength and nociception by the wire hang test (Figure 2) and hot plate test (Figure 3), respectively. It is possible that significant muscle weakness echoed and manifested the effects of less body weight gain in mice treated with ketamine for 6 months; less gain in body weight of 6-month ketamine treatment group compared to controls might indicate less muscle mass and strength leading to significantly poor performance in the wire hang and hot plate tests. In addition, it is possible that the motor nerve conduction velocity was reduced or damaged³⁹ by ketamine after 6 months' administration, potentially leading to impaired physical abilities. Further studies are necessary to elucidate the relationship and rationale between body weight, behavioral tests and physical performance impairments following long-term ketamine use.

Learning and working memory were not significantly affected, as in the water maze test results (Figure 4) for all 1-, 3- and 6-month groups, although the ketamine treatment groups consistently showed longer but not significant escape latency times than the respective control groups. Results of both the TUNEL and apoptotic markers (including Bax, Bcl-2 and caspase-3) showed no differences between the ketamine and control groups in the prefrontal cortex. Apparently, the function of prefrontal cortex with respect to learning and working memory did not appear permanently damaged, bearing in mind that we tested the mice 4 h after ketamine injection. However, other studies have shown significantly more apoptosis occurring in different brain regions, including the prefrontal cortex, in ketamine-administrated animals as compared to controls.^11,13,21,22 Water maze is generally considered to be a test of spatial learning and working memory,⁴⁰ but this might not always be the case because the performance of the mice could depend on other factors such as the use of different search strategies,⁴¹ fatigue⁴² and hypothermia.⁴³ Indeed, it has been shown that impairments in working memory induced by ketamine were not attributable to dysfunction of motivational, motor, short-term or spatial memory processes.²¹ Probably, further validation of the water maze test should be elucidated for more precise examination of the learning and working memory function of mice.²⁹

We deliberately performed the behavioral tests 4 h after ketamine injection because we were looking for stable and persistent damages in the brain caused by long-term ketamine rather than its immediate effects on the brain function of that particular daily injection. Ketamine is known to be short-acting and short-lived,^1,2 with no obvious outcomes even in cases of overdose.⁴ Most studies have examined the effects immediately after its injection.^21,22,44,45 In this study, we used a sub-anesthetic dose of 30 mg/kg, which is regarded as the recreational dose for rodents.⁴ In a recent unpublished pilot study, we experimented with different doses of ketamine at 30, 60 and 90 mg/kg. At 60 and 90 mg/kg of ketamine, there were mice fatalities after 1 month of ketamine injection. Also, ketamine was given only once daily to the mice, while in young human ketamine abusers, use of the drug would likely not be limited to only one time per day. Therefore, the significant and permanent damaging effects of ketamine on neuromuscular strength, nociception and possibly on body weight could be even more serious in human youngsters. This is a new finding and an important message illustrating that even at a reasonable recreational dose (one dose per day), the long-term use of ketamine is harmful to the brain and its functions, with definite permanent damage. This is a convincing message that could be used in anti-ketamine campaigns.

Using TUNEL and Western blot testing, we examined the prefrontal cortex for the presence of apoptotic lesions possibly caused by ketamine. There were no significant differences for all TUNEL and Western blot results between the ketamine and control groups; however, it may be worthwhile to note the consistent trends of higher Bax, lower Bcl-2, higher Bax/Bcl-2 and higher caspase-3 (to actin ratio; Table 1) showing the tendency of more apoptosis possibly associated with ketamine treatment. However, this does not exclude the possibility that the functions of prefrontal cortex may be affected without significant cellular damage. Indeed, it has been reported that in the rat, ketamine inhibited a subclass of fast spiking interneurons in prefrontal cortex that regulated the motivation of various behaviors.²² Hypofunction of the prefrontal cortex as induced by drugs could cause dysfunction leading to altered decision making by neglecting negative future consequences for immediate reward.⁴⁶ Another study showed that even without detectable cell death, ketamine produced aberrant diffuse network noise in multiple cortical and subcortical structures, including the prefrontal cortex, which might cause dysfunction of brain operations, including impairments in cognition and sensorimotor integration.⁴⁷ We have also made similar findings: a previous study by our group³³ reported significant increase of hyperphosphorylated tau in Layer I of the prefrontal cortex of mice treated with ketamine for 6 months. Layer I contains mainly communicating fibers; the presence of hyperphosphorylated tau in fibers would block the transmission through the fibers between cortical regions causing disruption of prefrontal cortex functions. Neuronal cell death may only occur secondarily with the eventual blockage of fiber transmission. Our previous results³³ also showed species differences: in monkey treated with ketamine (1 mg/kg) for 6 months, significant increases of hyperphosphorylated tau were found in neurons of the deeper layers of the prefrontal cortex. Such presence of hyperphosphorylated tau might signify immediate degeneration or death of the neurons themselves rather than the possible secondary cell damage or death as in mouse.

In our other series of experiments with long-term ketamine in mice (unpublished results), we found there was significant apoptosis in the cerebellum, suggesting there might be regional differences in response between different CNS regions in the mouse. In the present study, our results showed ketamine did not cause significant cellular damage or apoptosis in the prefrontal cortex, therefore further studies should examine brain function by in vivo or microdialysis methods,^22,48 comparing other regions of the brain outside the prefrontal cortex such as cerebellum. For instance, one direction of study on mechanisms of motor weakness due to ketamine could examine the known increase in glutamate release in nucleus accumbens in rat that leads to disruption of motor behavior and latent inhibition.⁴⁴

In conclusion, our research showed for the first time an important finding of ketamine use that 6 months of the usual recreational dose created stable and persistent damage in brain function. Despite no significant apoptosis detected in prefrontal cortex, young mice showed significant deterioration in neuromuscular strength and nociception following chronic use of ketamine. Further research is needed to determine the extent and mechanisms of potentially serious brain damage caused by this recreational drug.

Footnotes

Acknowledgement

We thank Ms E Lucy Forster for her critical review of the English of the manuscript.

This study was funded by the Grant from the Beat Drugs Fund Association, Hong Kong Government, Project Ref. No. 080048.

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

Abstract

Keywords

Introduction

Materials and methods

Experimental animals, groups and treatments

Behavioral studies

Wire hang test

Hot plate test

Water maze test

TUNEL assay

Western blot assay

Statistical method

Results

Body weight of mice

Behavioral studies

Wire hang test

Hot plate test

Water maze test

TUNEL and Western blot assay

Discussion

Footnotes

Acknowledgement

References