Regional and temporal profiles of calpain and caspase-3 activities in postnatal rat brain following repeated propofol administration

Abstract

Exposure of newborn rats to a variety of anesthetics has been shown to induce apoptotic neurodegeneration in the developing brain. We investigated the effect of the general anesthetic propofol on the brain of 7-day-old (P7) Wistar rats during the peak of synaptic growth. Caspase and calpain protease families most likely participate in neuronal cell death. Our objective was to examine regional and temporal patterns of caspase-3 and calpain activity following repeated propofol administration (20 mg/kg). P7 rats were exposed for 2, 4 or 6 h to propofol and killed 0, 4, 16 and 24 h after exposure. Relative caspase-3 and calpain activities were estimated by Western blot analysis of the proteolytic cleavage products of α-II-spectrin, protein kinase C and poly(ADP-ribose) polymerase 1. Caspase-3 activity and expression displayed a biphasic pattern of activation. Calpain activity changed in a region- and time-specific manner that was distinct from that observed for caspase-3. The time profile of calpain activity exhibited substrate specificity. Fluoro-Jade B staining revealed an immediate neurodegenerative response that was in direct relationship to the duration of anesthesia in the cortex and inversely related to the duration of anesthesia in the thalamus. At later post-treatment intervals, dead neurons were detected only in the thalamus 24 h following the 6-hour propofol exposure. Strong caspase-3 expression that was detected at 24 h was not followed by cell death after 2- and 4-hour exposures to propofol. These results revealed complex patterns of caspase-3 and calpain activities following prolonged propofol anesthesia and suggest that both are a manifestation of propofol neurotoxicity at a critical developmental stage.

Fig. 1

Timeline of the experiments. Each rectangle marks a 2-hour time period, the gray color represents the duration of the anesthesia. Time point 0 indicates termination of anesthesia. Small arrows point to propofol injections (20 mg/kg), and the arrowheads indicate the postexposure intervals at which the animals were killed.

Fig. 2

Time course of spectrin breakdown products after the 2-, 4- and 6-hour exposures to propofol in the cortex and thalamus of P7 rats. Whole-cell extracts were used to detect the presence of the 280-kDa intact spectrin protein; calpain produced a 145-kDa fragment and caspase-3 produced a 120-kDa fragment in the cortex (a) and the thalamus (b). Bars represent a quantitative densitometric evaluation of the 145-kDa spectrin fragment in the cortex (c) and the thalamus (d), and the 120-kDa spectrin fragment in the cortex (e) and the thalamus (f). Results are presented for animals at different recovery time points (0, 4, 16 and 24 h) after exposure to propofol for 2, 4 and 6 h. Representative immunoblots of the 4-hour treatment are shown. β-Actin was run as an internal standard for equal loading. The results are the means ± SEM. ^a p < 0.05 vs. control value presented as a black line, ^b p < 0.05 between treatments.

Fig. 3

Time course of appearance of PARP-1 cleavage fragments in the cortex and thalamus of P7 rats after exposure to propofol for 2, 4 and 6 h. Whole-cell extracts were used to detect the presence of 50-, 40- and 20-kDa PARP-1 fragments in the cortex (a) and the thalamus (b). Bars represent quantitative densitometric evaluation of the approximately 50-kDa PARP-1 fragment in the cortex (c) and the thalamus (d), the approximately 40-kDa PARP- 1 fragment in the cortex (e) and the thalamus (f), and the approximately 20-kDa PARP-1 fragment in the cortex (g) and the thalamus (h). Results are presented for animals at different recovery time points (0, 4, 16 and 24 h) after exposure to propofol for 2, 4 and 6 h. Representative immunoblots of the 2-hour treatment are shown. β-Actin was run as an internal standard for equal loading. Results are presented as means ± SEM. ^a p < 0.05 vs. control value presented as a black line, ^b p < 0.05 between treatments.

Fig. 4

Time course of PKC levels in the cortex and thalamus of P7 rats after 2-, 4- and 6-hour exposures to propofol. Whole-cell extracts were used to detect the presence of the 82-kDa intact PKC protein and the calpain-produced 36-kDa PKC fragment in the cortex (a) and the thalamus (b). Bars represent quantitative densitometric evaluation of the intact 36-kDa protein in the cortex (c) and the thalamus (d). Results are presented for animals at different recovery time points (0, 4, 16 and 24 h) after exposure to propofol for 2, 4 and 6 h. Representative immunoblots of the 6-hour treatment are shown. β-Actin was run as an internal standard for equal loading. The results are the means ± SEM. ^a p < 0.05 vs. control value presented as a black line, ^b p < 0.05 between treatments.

Fig. 5

Time course of appearance of active caspase-3 subunits in the cortex and thalamus of P7 rats after exposure to propofol for 2, 4 and 6 h. Whole-cell extracts were used to detect the appearance of active 19/17-kDa caspase-3 subunits in the cortex (a) and the thalamus (b). Bars represent quantitative densitometric evaluation of 19/17-kDa subunits in the cortex (c) and the thalamus (d). Results are presented for animals at different recovery time points (0, 4, 16 and 24 h) after exposure to propofol for 2, 4 and 6 h. Representative immunoblots are shown. β-Actin was run as an internal standard for equal loading. Results are presented as means ± SEM. ^a 0 < p.05 vs. control value presented as a black line, ^b p < 0.05 between treatments.

Fig. 6

Fluoro-Jade B staining of P7 rat brains immediately (0 h) after termination of the 2-, 4- and 6-hour exposures to propofol. An increase in the number of Fluoro- Jade B-positive cells was apparent in the retrosplenial cortex following 4 and 6 h of exposure (c, d, left panel), and in the laterodorsal thalamic nucleus following 2-, 4- and 6-hour exposure (b, c, d, respectively, right panel). Quantitative analysis of Fluoro-Jade B staining at the retrosplenial cortex and the laterodorsal thalamic nucleus is shown (e). For each condition, 3 animals were randomly assigned. Data are presented as means ± SEM. A probability of * 0 < p.05 was considered significant. Degenerating neurons are marked with arrows, arrowheads point to blood vessels. Scale bar = 20 μm.

Fig. 7

Fluoro-Jade B staining of P7 rat brains at the 24-hour time point following exposure to propofol for 6 h. An increase in the number of Fluoro-Jade B-positive cells was apparent in the thalamus (b) but not in the cortex (a). c The laterodorsal thalamic nucleus was stained with Hoechst 33258, a blue fluorescent DNA stain, alongside Fluoro-Jade B. Some neurons were double-labeled; the same neuron revealed bright clumps of fragmented and condensed chromatin, typical for apoptotic nuclei, as well as positive green staining for Fluoro-Jade B (×40). Degenerating neurons are marked with arrows, arrowheads point to blood vessels. Scale bar = 20 μm.