* Division of Neurotoxicology, NCTR/FDA, Jefferson, Arkansas 72079; USUHS, Bethesda, Maryland 20892;
CDER/FDA, Silver Spring, Maryland 20993; and
NIDA/NIH, Bethesda, Maryland 20892
Received May 27, 2004; accepted July 9, 2004
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ABSTRACT |
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Key Words: neurodegeneration; apoptosis; blood levels; anesthesia; NMDA; caspase-3.
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INTRODUCTION |
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Recently, NMDA antagonists, whether given alone or in combination with GABA (gamma amino butyric acid) agonists, markedly increased neurodegeneration throughout the developing brain of seven-day-old rat pups (Ikonomidou et al., 1999, 2001
; Ishimaru et al., 1999
; Olney et al., 2000
, 2002a
,b
,c
). As observed for other NMDA antagonists such as MK-801 (dizocilpine, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine), ketamine produced elevated numbers of apoptotic neurons throughout the seven-day-old rat brain (Ikonomidou et al., 1999
). However, while MK-801 neurotoxicity occurred after a single exposure, ketamine neurotoxicity was reported only after a regimen of multiple injections given during a period of about 9 h (Ikonomidou et al., 1999
). The brain region most affected by ketamine was the dorsolateral thalamus, where ketamine-treated animals had a 31-fold increase in neurons stained selectively for neurodegeneration with the De Olmos silver method. These results suggested that NMDA receptor stimulation was a critical factor for neuronal survival during development and that suppression of this activity resulted in an increase in apoptotic cell death (Haberny et al., 2002
; Olney, 2002
; Olney et al., 2002c
).
The present study sought to confirm the finding that multiple doses of ketamine administered over a short period of time to the early postnatal rat pup would result in widespread increases in neurodegeneration, using the same procedures (Ikonomidou et al., 1999). We also wished to include an independent measure of neurodegeneration, the green fluorescent stain Fluoro-Jade B (Schmued and Hopkins, 2000
; Ye et al., 2001
), which, unlike silver methods, can be used in multiple staining procedures for cellular apoptotic markers. Finally, we wanted to include lower doses and briefer exposures to ketamine, so that we could begin to characterize the blood levels of ketamine associated with its neurotoxicity in perinatal rat pups.
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MATERIALS AND METHODS |
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Blood collection. Rat pups from each group (n = 34) were sacrificed by decapitation 2 min after their last injection and their blood was collected for analysis of ketamine levels by high performance liquid chromatography (HPLC) using modified literature methods (Adams et al., 1992; Gross et al., 1999
). The plasma was isolated from whole blood by centrifugation at 4°C. The plasma supernate was transferred to plastic cryovials, placed on dry ice until frozen, and then stored in a 80°C freezer until analysis. Gender was determined at the time of sacrifice.
Sample preparation. Analytes were extracted from rat plasma after application to silica based solid phase extraction (SPE) C-2 cartridges using a modification of a literature SPE method (Mistry et al., 1998). Ketamine and bupivacaine, the internal standard (IS), were added to the plasma samples. Plasma samples and standards (500 ul) were diluted 1:1 with water and vortexed for 30 s. The 200 mg C-2 SPE columns (Varian, Harbor City, CA) were conditioned with (1) 3 ml of methanol (MeOH) and (2) 3 ml of water. Plasma samples were loaded onto the SPE column then rinsed with (1) 3 ml of water and (2) 3 ml of 50:50 MeOH/water. Samples were eluted with 50:50 acetonitrile/0.2 N hydrochloric acid (HCl). Samples were dried under vacuum in a spin evaporator (Savant, Farmingdale, NY) and reconstituted in 150 µl of the mobile phase.
Plasma analysis. All reconstituted standards and samples were analyzed on a Hewlett-Packard 1090 (Wilmington, DE) HPLC system equipped with a multi-channel pump, auto-injector, solvent degasser, and diode array detector (DAD). Separation was achieved on a Phenomenex C-18 Luna (2), 5 micron (250 x 4.6 mm) reverse phase HPLC column (Torrance, CA) with a Phenomenex C-18 guard cartridge (4.0 x 3.0 mm). The mobile phase was acetonitrile/10 mM phosphate, pH = 3.0 (11:89, v/v) delivered isocratically for 22 min. The flow rate was 1.0 ml/min. The injection volume for samples and standards was 50 µl. The UV detection wavelength was 205 nm. The observed retention times for ketamine and the internal standard bupivacaine were 9.9 and 15.9 min respectively.
Chemicals and reagents. Ketamine and bupivacaine were purchased from Sigma (St. Louis, MO). Acetonitrile and methanol HPLC grade were purchased from Burdick & Jackson (Muskegon, MI). HPLC grade monobasic potassium phosphate and ACS grade hydrochloric acid were purchased from Fisher Scientific (Pittsburgh, PA). Filtered 18 meg-ohm water was supplied in-house by a Millipore Milli-Q System (Bedford, MA). Blank rat plasma was purchased from Hilltop Laboratories (Scottsdale, PA).
Perfusion. The remaining rat pups (those not sacrificed for blood levels of ketamine) were deeply anaesthetized with sodium pentobarbital 24 h after the initial dose (i.e., on PND 8), They were then perfused transcardially, with a saline flush (about 10 ml) followed by about 50 ml of 4% paraformaldehyde fixative in neutral cacodylate buffer. Their brains were then removed and stored in fixative until further processing.
Histological processing. The rat pup brains were then equilibrated with sucrose and embedded in gelatin as batches of 16 brains with all of the groups represented in each batch. They were then frozen prior to serial sectioning on a sledge microtome. Sets of 16 coronal sections, one from each of the 16 brains and each from approximately the same anteroposterior plane of section were then mounted on 2 x 3 inch glass slides. A one in five series of these slides were then stained with a cupric silver method selective for neurodegeneration (Beltramino et al., 1993).
Certain of the remaining sections were then used for triple fluorescent staining of (1) immunoreactive caspase-3, (using a rabbit polyclonal antisera directed against the active 18 kDa form of caspase-3, Trevigen, Gaithersburg, MD) (Scallet, 1995; Scallet et al., 1988
), (2) degenerating neurons using FluoroJade-B (Schmued and Hopkins, 2000
; Ye et al., 2001
), and (3) nuclear DNA using DAPI (4',6-diamidino-2-phenylindole) (Spackova et al., 2003). Briefly, the procedure involved rinsing the sections three times for 5 min each in 0.1 M phosphate buffer (pH 7.4), followed by an overnight incubation in anti-caspase-3 antisera at 5°C. The primary rabbit anti-caspase-3 antisera was diluted 1:500 in an antibody diluent prepared with 2% normal goat serum and 0.3% Triton X-100 in 0.1 M phosphate buffer. The next day, following three more 5 min rinses in buffer, sections were incubated for an hour in a 1:100 dilution in antibody diluent of the secondary antibody, a goat anti-rabbit antisera conjugated with rhodamine (Chemicon, Temecula, CA). Following three more rinses in buffer, the sections were viewed wet under green excitation for red rhodamine epifluorescence to confirm the labeling of caspase-3 immunopositive apoptotic neurons in the laterodorsal thalamus. Then the sections were rinsed in water for about 2 min and in potassium permanganate (0.06%) for 10 min with mild agitation. Following another 2 min rinse in water, the sections were incubated in 0.1% acetic acid containing a mixture of 0.0002% DAPI (Sigma Chemical, St. Louis, MO) and 0.0004% Fluoro-Jade B (Histo-Chem, Jefferson, AR). The resulting sections were then viewed individually and photographed with epifluorescence using green incident light for caspase-3, ultraviolet light for DAPI, and blue light for Fluoro-Jade B.
Morphometric methods. The dosolateral thalamus was previously reported to have sustained the largest fold-increase in degenerating neurons following ketamine exposure (Ikonomidou et al., 1999). Thus the dorsolateral thalamus, with the addition of the medial amygdala, were selected for the purpose of morphometric confirmation of the previous findings (Ikonomidou et al., 1999
).
Slides were selected that contained the Lateral Dorsal thalamus, VentroLateral division (LDVL according to the rat brain atlas) at an anteroposterior level of about 2.5 mm from bregma (Paxinos and Watson, 1986). Because the rat brains were embedded in register, usually a single slide contained sections taken from the same anteroposterior level from about 12 of the 16 brains represented on each slide. If a given brain's sections were a little more anterior or posterior than the others, it was a simple matter to select the preceeding or succeeding slide in order to sample the LDVL and the medial amygdala at a comparable anteroposterior level for each brain.
The silver degeneration procedure produced high-contrast staining of dark positive neurons against a light background (see Figs. 1b and 1d). Using an image analysis system (MCID5 +, Imaging Research, Inc., St. Catherine's, Ontario, Canada), two investigators unaware of the treatment conditions agreed on an outline of the LDVL and medial amygdala from each brain. The area of the outlined brain nucleus was then provided by the image analysis system. The greyscale thresholds were set midway between the mean signal intensity of a positive neuron and the mean background level for each brain section in order to systematically segment positive neurons from background and provide a total count as previously described (Scallet et al., 2000). Data were then expressed as the mean frequency of degenerating, silver-positive cells per mm2 of the brain region and as fold-increases for purposes of comparison with the original report on ketamine neurotoxicity (Ikonomidou et al., 1999
).
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RESULTS |
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Ketamine exposure also increased the number of medial amygdalar neurons positively stained for neurodegeneration (F(3,32) = 5.9, p < 0.01; see Figure 1f for quantitative comparisons between groups and see Figures 1d vs. 1c for appearance). Tukey Multiple Comparisons indicated that the "high dose" group (seven repeated doses of 20 mg/kg ketamine) was significantly greater (p values < 0.05) than each of the other three groups (control, low, and high single), which were not significantly different from each other. The elevation of the "high dose" group, while statistically significant, represents only about a three-fold increase over control levels of degenerating neurons. Apparently, the numerically smaller fold-increase is because the control levels of degenerating neurons in the medial amygdala were higher than in the LDVL despite comparable levels between the two regions in the "high dose" group (see Figure 1e vs. 1f).
Fluoro-Jade B vs. DeOlmos Silver Neurodegeneration Staining in the LDVL
To compare Silver and Fluoro-Jade staining results, examine Figures 2a, 2b, 2c, and 2d. Figure 2a is a silver-stained control, easily distinguished from Figure 2b, a silver-stained section from a ketamine-treated rat pup. Figure 2c is a Fluoro-Jade-stained control of comparable appearance to the silver-stained control of Figure 2a, while the Fluoro-Jade-stained degenerating "treated" neurons in Figure 2d are quite comparable both in number and pattern to the silver-stained neurons of Figure 2b. Figure 2f is a silver-stained section from the hippocampus of a ketamine-treated rat, while Figure 2e is a nearly adjacent section from the same animal, but stained with Fluoro-Jade-B.
Multiple Fluorescent Labeling of Neurons in "High Dose" Ketamine-Exposed Rat Pups
To further investigate the mode of death exhibited by these neurons, coronal sections were stained with Fluoro-Jade B (FJ-B, a green fluorescent marker of neurodegeneration, as well as DAPI (4', 6-diamidino-2-phenylindole dihydrochloride, a blue fluorescent DNA stain) and an antibody to the apoptotic marker, caspase-3 (CASP, labeled red with tetramethylrhodamine fluorescence).
The results from the high dose subjects (see Fig. 2) reveal that many cells in the LDVL thalamus contained one or more foci of compact, bright DAPI-stained chromatin (Fig. 2g), an indication that they were undergoing apoptosis. Most such neurons also stained positively with both FJ-B (Fig. 2h) and with the anti-CASP antiserum (Fig. 2i), although some neurons positive for both CASP and FJ-B were not DAPI-positive for apoptosis.
Blood Levels of Ketamine
Table 1 indicates that the blood levels achieved by the repeated 20 mg/kg dosing protocol are considerably higher (around 14 mg/ml) than the blood levels in pups receiving either the multiple 10 mg/kg doses or a single dose of 20 mg/kg (each around 25 mg/ml).
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DISCUSSION |
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The seven repeated doses of 20 mg/kg ketamine resulted in a 28-fold increase in the number of degenerating neurons within the laterodorsal thalamic nucleus of the rat pups. The measured increase in the present study compares very favorably with the 31-fold increase of degenerating neurons in the same region of the thalamus as reported by Ikonomidou et al. (1999). While that original report is clearly replicated by the present research, we also observed that seven repeated smaller (10 mg/kg) doses were without measurable effect. Also, a single dose of 20 mg/kg was ineffective at producing neurodegeneration.
Blood was drawn from groups of rats sacrificed on PND 7, 2 min after their last injection. The blood levels of ketamine associated with ineffective exposures were 25 µg/ml while the level associated with a neurotoxic exposure was about 14 µg/ml. An anesthetic blood level in humans is about 2 µg/ml (Malinovsky et al., 1996; Mueller and Hunt, 1998
), suggesting the possibility that a prolonged supra-anesthetic dose may be needed for neurotoxicity. Our animals at the repeated 10 mg/kg and single 20 mg/kg dose levels appeared anesthetized, but no actual tests of their level of analgesia or anesthesia were conducted. The fact that seven multiple 20 mg/kg doses produced higher blood levels of ketamine than a single 20 mg/kg dose suggests that repeating the injections every 90 min allowed the ketamine to accumulate in blood and/or tissue. The elimination half-life of ketamine in humans is around 2.5 h (12 h in a pediatric population) (Domino et al., 1997
; Hartvig et al., 1993
), so accumulation in tissue of ketamine from doses separated by only 90 min seems possible. Future work focused on the precise ketamine exposure parameters required to produce analgesia, anesthesia, and neurotoxicity will prove helpful.
Another goal of this research was to use a multiple staining approach to more reliably identify the dying cells as undergoing apoptosis (Dikranian et al., 2001). Caspase-3 is a cytoplasmic protein which can be activated by cleavage, initiated either by the stimulation of TNF (tumor necrosis factor) receptors on the surface of the membrane or by the release of cytochrome c from mitochondria undergoing calcium-activated pore transition. Following cleavage into an active 18 KDa fragment, caspase-3 is involved as an effector protein degrading downstream targets including structural proteins, inhibitors of apoptosis, DNA repair enzymes, cell cycle proteins, and signal transduction molecules which can then result in the occurrence of apoptosis (Ananth et al., 2001
; McLaughlin, 2004
; Vyas et al., 2002
; Wang, 2000
; Yuan et al., 2003
). The subsequent research on NMDA antagonist-induced neurotoxicity has identified the presence of apoptotic neurons by electron microscopy and by the use of specific antisera directed against caspase-3 (Olney et al., 2002a
). However, the various types of labeled cells noted to respond to NMDA antagonist exposure had to be evaluated on, at best, adjacent brain sections taken from similar regions.
Here, we showed that Fluoro-Jade B could identify apoptotic cells produced after exposure to the NMDA antagonist ketamine. Then, we used Fluoro-Jade B to stain degenerating cells in sections that were also stained for chromatin and caspase-3. We were able to show that in many cases the degenerating cells were triple-labeled; the same neuron revealed bright clumps of DAPI-positive chromatin characteristic of apoptotic nuclei, as well as positive green staining for Fluoro-Jade B and red for caspase-3. However, there were some DAPI-negative neurons that nevertheless stained positively for both Fluoro-Jade B and caspase-3 and a few that appeared to be positive for Fluoro-Jade B but not for caspase-3. Probably, the stains are differentially sensitive to the various stages that apoptotic cells undergo; further studies should be undertaken with cultured cells, where the occurrence of apoptosis can be synchronized following a toxicant exposure.
The present study confirms previous research indicating the neurotoxicity of ketamine, demonstrates multiple staining approaches for a single section that may be used to identify different stages of apoptosis, and suggests that neurotoxic blood levels of ketamine may be somewhat higher than levels required for anesthesia. Further research will be necessary to resolve concerns regarding the doses and durations of ketamine exposure that are safe for pediatric populations.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at the Division of Neurotoxicology, National Center for Toxicological Research, 3900 NCTR Drive, Jefferson, AR 72079. Fax: (870) 543-7745. E-mail: AScallet{at}nctr.fda.gov.
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