Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain

Abstract

Recently, it was reported that anesthetizing infant rats for 6 h with a combination of anesthetic drugs (midazolam, nitrous oxide, isoflurane) caused widespread apoptotic neurodegeneration in the developing brain, followed by lifelong cognitive deficits. It has also been reported that ketamine triggers neuroapoptosis in the infant rat brain if administered repeatedly over a period of 9 h. The question arises whether less extreme exposure to anesthetic drugs can also trigger neuroapoptosis in the developing brain. To address this question we administered ketamine, midazolam or ketamine plus midazolam subcutaneously at various doses to infant mice and evaluated the rate of neuroapoptosis in various brain regions following either saline or these various drug treatments. Each drug was administered as a single one-time injection in a dose range that would be considered subanesthetic, and the brains were evaluated by unbiased stereology methods 5 h following drug treatment. Neuroapoptosis was detected by immunohistochemical staining for activated caspase-3. It was found that either ketamine or midazolam caused a dose-dependent, statistically significant increase in the rate of neuroapoptosis, and the two drugs combined caused a greater increase than either drug alone. The apoptotic nature of the neurodegenerative reaction was confirmed by electron microscopy. We conclude that relatively mild exposure to ketamine, midazolam or a combination of these drugs can trigger apoptotic neurodegeneration in the developing mouse brain.

Figure 1

(a) Neuronal profiles showing caspase-3 activation (C3A) in the neocortex (layer II) and caudate-putamen of the 7-day-old mouse brain 5 h after a single subcutaneous injection of saline or ketamine, 40 mg kg⁻¹. In the neocortex and caudate-putamen of the saline control pup, there are a few scattered C3A-positive neuronal profiles, reflecting the normal rate of physiological cell death at this developmental age. In the same brain regions of the ketamine-treated pup, the number of C3A-positive profiles is several-fold increased, consistent with the quantitative data presented in Table 2. Extreme sensitivity of layer II neocortical neurons, as shown here for ketamine, has been reported previously (Ikonomidou et al., 1999; 2000) as a feature of the apoptogenic properties of other NMDA antagonists, including MK-801, phencyclidine and ethanol. Scale bar=50 μm. (b) The apoptosis-promoting effect of ketamine is dose-dependent. At 5 h after littermates were treated with saline or ketamine, 10, 20, or 30 mg kg⁻¹, the C3A positive profiles in the caudate-putamen region showed a linear dose-dependent increase (test for linear trend: P=0.0004, r²=0.1288). Both ketamine 20 mg kg⁻¹ (P< 0.05) and 30 mg kg⁻¹ (P<0.01) induced a significant increase in C3A positive profiles compared to their litter-matched controls (n=10 for each treatment group, P=0.0038, F (3, 9, 27)=5.671, repeated measures ANOVA with Student–Newman–Keuls multiple comparisons test, comparing saline,with ketamine 10, 20, and 30 mg kg⁻¹). When the data for Ketamine 40 mg kg⁻¹ (from Table 2) are displayed on the same graph, it is seen that, whereas the rate of apoptosis increased approximately 23% with each stepwise increase in dose in the zero to 30 mg kg⁻¹ dose range, the rate of increase jumped by 100% as the dose was increased from 30 to 40 mg kg⁻¹.

Figure 2

Brains of 7-day-old mice treated with midazolam 9 mg kg⁻¹ (a, c) or midazolam plus ketamine 40 mg kg⁻¹ (b, d) showed robust C3A staining in cerebral cortex (c, d) and caudate-putamen region (a, b) 5 h after treatment. The combined treatment of midazolam plus ketamine was more damaging in both regions than midazolam alone.

Figure 3

Quantitative analysis of C3A profile density revealed that combined midazolam and ketamine treatment was more effective in causing neuronal apoptosis than either drug alone. A total of 12 litters of pups were used for this analysis. (a) In caudate-putamen, ketamine plus midazolam produced a C3A profile density of 35.7±4.5 profile mm⁻² (n=20), significantly higher than that of the ketamine group (23.2±3.1 profile mm⁻², n=17, P<0.05) or midazolam group (21.6±3.2 profile mm⁻², n=21, P<0.05). One-way ANOVA with Student–Newman–Keuls test, P=0.0162, F (2, 55)=4.447. (b) In the cerebral cortex, the C3A profile density for the ketamine plus midazolam group was 16.0±1.0 profile mm⁻² (n=20), significantly higher than that of the ketamine group (7.6±0.6 profile mm⁻², n=17, P<0.001) or midazolam group (10.0±4.6 profile mm⁻², n=21, P<0.001). One-way ANOVA with Student–Newman–Keuls test, P<0.0001, F (2, 55)=21.209.

Figure 4

Sections stained by the De Olmos silver method from the neocortex (layer II) and caudate-putamen of the 7-day-old mouse brain 7 h after a single subcutaneous injection of saline or ketamine, 40 mg kg⁻¹. The silver stain, which marks neurons that are dead or dying, reveals the same pattern of neurodegeneration that was observed in C3A-stained sections in Figure 1. At 7 h post-treatment, the affected neurons are in a more advanced stage of degeneration and, therefore, have a more condensed and fragmented appearance. Scale bar=75 μm.

Figure 5

Ultrastructural appearance of a normal neocortical neuron (a), a neocortical neuron undergoing hypoxia/ischemia-induced acute excitotoxic degeneration (b) and neurons in the neocortex (c) or caudate/putamen (d) undergoing acute apoptotic degeneration 5 h following exposure to ketamine. All photos are from the 7-day-old mouse brain. The earliest changes in excitotoxic neurodegeneration (b) are swelling and disintegration of cytoplasmic constituents, primarily mitochondria and endoplasmic reticulum. This is followed by formation of multiple small clumps of nuclear chromatin that distribute around the perimeter of the nucleus in a clockface configuration. These clumps then coalesce into larger irregularly shaped chromatin masses (large arrow) that migrate to the center of the nucleus, causing the nucleus to appear pyknotic. In contrast to hypoxic/ischemic excitotoxic changes, ketamine exposure causes developing neurons to display a sequence of classical changes characteristic of apoptosis. The earliest changes associated with apoptosis are relatively inconspicuous mitochondrial defects (MD) in the cytoplasm and very conspicuous formation of large spherical chromatin balls (CB) in the nucleus. These early changes are followed by fragmentation or disappearance of the nuclear membrane (thin arrow), disaggregation of the nucleolus into granular (G) and filamentous (F) components and condensation of the entire cell. The changes associated with hypoxic/ischemic neurodegeneration and apoptotic neurodegeneration are different both in nature and in sequence. Scale bar=1 μm.