10.1007/s12640-009-9063-8
Materials and Methods
The experiments were performed according to the guidelines of the German Animal Protection Law and were approved by the Berlin State authorities.

Wistar rat pups were purchased from the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin BgVV, Berlin, Germany.

Experimental Protocol
Six-day-old Wistar rats received either intraperitoneal (i.p.) injections of propofol or underwent inhalational anesthesia with sevoflurane and were separated from their mother during the experimental phase. In every litter animals were randomized either for anesthesia or controls.

For propofol anesthesia doses of 30 mg/kg body weight were given every 90 min up to a cumulative dose of 90 mg/kg. For gas administration rats were placed into an incubation chamber (Billups Rothenberg Inc., Del Mar, USA), which was connected to an anesthesia system (F.Stephan GmBH, Gackenbach) for 6 h. Carbon dioxide and sevoflurane concentrations were monitored using a gas monitor (Datex Ohmeda, GE Healthcare, Munich, Germany). To avoid rebreathing of carbon dioxide, inspired CO2 concentration was continuously monitored and kept below 1 vol% by adjusting the fresh gas flow. Pilot studies established that sevoflurane concentrations between 3 and 5% maintained sufficient depth of anesthesia, as determined by lack of reaction to a painful stimulus. However, it was also observed that skin color changed and respiratory diminished in some animals, suggesting respiratory insufficiency. Accordingly, animals were closely monitored during the experiment and sevoflurane concentration adjusted between 3 and 5 vol% to maintain normal skin color and adequate respiratory efforts.

The animals were observed for another 90 min until they were awake and active to be returned to their mother after the last injection and 6 h of gas application respectively. To maintain body temperature and prevent hypothermia, animals were placed on a heating device. During anesthesia respiratory frequency and skin color were observed to detect apnea and hypoxia. If bradypnoe occurred, rats received a pain stimulus, if breathing did not restart or resuscitation efforts were necessary rats were excluded from further processing and analysis. Verum and control animals received injections of PÄD II cristalloid/glucose (50 g/l) solution (Fresenius-Kabi, Bad Homburg, Germany) to prevent hypoglycemia and hypovolemia. Control animals were separated from the mother for the same period as the anesthetized animals and received injections of the crystalloid/glucose solution as well. In order to reduce the amount of laboratory animals, the control animals of the propofol group were pooled with control animals from the sevoflurane group. Therefore, sham injections have not been performed. However, all experiments were performed using the same experimental settings and laboratories during the same time.

For perfusion fixation animals were killed with an injection of an overdose of chloral hydrate 24 h after starting anesthesia. A solution of PBS (phosphate buffered saline) mixed with heparine (Thrombophob 25,000, Heparin-Natrium; Nordmark Arzneimittel GmbH, Uetersen, Germany) was injected slowly into the heart and ascending aorta in order to wash out the blood from the vessels. Afterwards, rats were perfused with a solution containing paraformaldehyde 4% (Merck, Darmstadt, Germany) with cacodylate buffer (Sigma, Deisenhofen, Germany) for 10 min (De Olmos cupric silver staining).

Histology
To visualize degenerating cells, coronal sections (70 μm) of the whole brain were cut on a vibratome and stained with silver nitrate and cupric nitrate (De Olmos and Ingram 1971). This technique stains degenerating cells dying via an apoptotic or non-apoptotic mechanism. Degenerating cells were identified by their distinct dark appearance due to silver impregnation.

Quantification of Damage
Quantification of brain damage was assessed in the frontal, parietal, cingulate, retrosplenial cortex, caudate nucleus (mediodorsal part), thalamus (laterodorsal, mediodorsal, and ventral nuclei), septum, dentate gyrus, hypothalamus, cornu ammonis field CA1, and subiculum in silver stained sections by estimating mean numerical densities (Nv) of degenerating cells (Gundersen et al. 1988). An unbiased counting frame (0.05 mm × 0.05 mm: dissector height 0.07 mm) and a high aperture objective were used for sampling. The Nv for each brain region was determined with 8–10 dissectors. Regional Nv values from 17 brain regions were summed to give a total score for degenerating neurons for each brain. Figure 1 shows representative silver stained brain regions.

Fig. 1
figure 1
Light microscopic overviews of silver-stained transverse sections picturing neurodegenerative changes in 6-day-old rats. The images of the rat thalamus show examples 24 h after treatment a after propofol treatment, b sevoflurane treatment, and c for controls. Degenerated neurons are pictured as small dark dots

Full size image
Behavioral Task
For behavioral testing n = 13 propofol treated, n = 7 sevoflurane treated animals and n = 6 controls were anesthetized as described above. Only male pups were used. At the beginning of behavioral testing, animals were 7 weeks old. Animals were group-housed (4–5 animals per cage) under standard laboratory conditions (22 ± 2°C room temperature) with an artificial 12 h light-dark cycle (lights on 6.00–18.00 h). Rats had access to food (Altromin 1326, Lage, Germany) and water ad libitum. They were acclimated to the animal unit for at least 2 weeks before testing. One hour prior to testing rats were transferred to a quiet anteroom. All experiments were performed between 8.00 and 13.00 h and only male animals were tested. In order to avoid possible carry-over effects, a pause of 7 days, during which the animals were left undisturbed in their home cages, was introduced between the hole board and water maze task.

Morris Water Maze (MWM) Test
The water maze task was conducted by using the same experimental design as described previously (Bert et al. 2002). A blue circular tank (diameter 200 cm, 60 cm deep) was filled up with water (21 ± 1°C) to a height of 42 cm. The tank was surrounded by several visual cues and was indirectly illuminated (120 lx at the centre of the pool).

For a single adaptation trial (day 0, without platform), the rats were released into the pool for 90 s with no escape platform present. On the following 8 days (day 1–8, place version) a transparent platform (16 × 16 cm) was submerged 1.5 cm below the surface in the middle of one of four virtual quadrants which was according to adaptation trial neither preferred nor avoided by the rat. Each day the animals were lowered into the water facing the wall from three different starting points (left, opposite, and right from the platform quadrant). Animals that did not find the escape platform within 90 s were placed onto it by the experimenter. All rats were allowed to remain on the platform for 30 s for orientation and were afterwards removed to rest for 60 s in a heated cage until the next trial. For each trial the escape latency to reach the platform was measured by a computerized tracking system (TSE VideoMot, Version 1.43, Bad Homburg, Germany). For each animal the three daily trials were averaged. On day 9 the escape platform was removed (spatial probe) and the time spent in each quadrant during a single 90 s trial was registered. On day 10 (cued version) the platform was elevated 1 cm above water level, signaled by a white cylinder (diameter 3 cm and 4 cm high), and moved to the quadrant opposite to the initial quadrant. This test was performed to assess the motivation to escape from the water and sensor-motor integrity. The testing procedure and recorded parameter during the cued version were the same as for the hidden platform version of the task (Morris 1984).

Hole Board Test
The hole board apparatus consisted of a square box (50 × 50 cm) made of grey Perspex with 16 equally spaced holes (diameter 2.5 cm), and was situated in a sound-attenuated chamber. The behavior of rats was monitored by an overhead installed video camera which was linked to a computerized tracking system (TSE VideoMot2, Bad Homburg, Germany). The test was conducted on two consecutive days. On both days rats were placed in the centre of the apparatus and observed for 10 min. The numbers of nose pokes and rearings as well as the distance traveled were recorded. After each animal the box was cleaned with 2-propanol 30%. Habituation to the apparatus was defined as a significant reduction of nose pokes, rearings and locomotor activity from the 1st to the 2nd day (Voits et al. 1995).

Blood Gas Analysis
To exclude severe hypoxia, hypercapnia or lactic acidosis, a blood gas analysis was performed for example in one animal of each group by transcutaneous puncture of the left ventricle. The probe was analyzed by a blood gas analyzer (Radiometer ABL series, Radiometer, Copenhagen, Denmark).

Statistical Analysis
The Kolmogorov Smirnov test was used to test for normal distribution. The results of the sum scores were compared using the Mann–Whitney U test between controls and propofol as well as between controls and sevoflurane. The place version data of the water maze test were analyzed by two-way ANOVAs on repeated measures followed by Holm-Sidak method for post-hoc multiple pair-wise comparisons. The spatial probe and the cued version data were analyzed by one-way ANOVAs followed by Holm-Sidak post-hoc tests. The data of the hole board test were analyzed by paired t-tests. Differences were considered to be significant if P < 0.05.