Original Article Efficacy of rutin in inhibiting neuronal apoptosis … · 2018. 8. 31. · Abstract: Sevoflurane and propofol are widely used in pediatric anesthesia. Neurotoxicity

Int J Clin Exp Med 2015;8(8):14397-14409www.ijcem.com /ISSN:1940-5901/IJCEM0009873

Original ArticleEfficacy of rutin in inhibiting neuronal apoptosis and cognitive disturbances in sevoflurane or propofol exposed neonatal mice

Yi-Gang Man, Rui-Gang Zhou, Bing Zhao

Department of Children’s Nervous and Rehabilitation, Jining No. 1 People’s Hospital, Jining 272111, Shandong, China

Received May 4, 2014; Accepted August 3, 2015; Epub August 15, 2015; Published August 30, 2015

Abstract: Sevoflurane and propofol are widely used in pediatric anesthesia. Neurotoxicity of sevoflurane and propo-fol in developing brain has been reported and these effects raise concerns on the usage of the drugs. We investigat-ed the influence of rutin, a flavonoid on the neurodegenerative effects of sevoflurane and propofol and on memory and cognition in neonatal rodent model. Separate groups of neonatal mice (C57BL/6) were administered with rutin at 25 or 50 mg/kg body weight (b.wt) from post natal day 2 (P1) to P21. P7 mice were exposed to 2.9% sevoflurane and/or propofol (150 mg/kg b.wt). Neuroapoptosis was assessed by measuring activated caspase-3 and by Fluoro-Jade C staining. Plasma S100β levels were detected by ELISA. Morris water maze test was performed to test learning and memory impairments in the animals. General behaviour of the mice was also assessed. Anesthesia exposure caused severe neuroapoptosis and also raised the levels of plasma S100β. Neuroapoptosis, memory and cognitive deficits observed following anesthetics were comparatively more profound in mice on exposure to combined drug (sevoflurane and propofol) than in those exposed to either of the anesthetics. Rutin at both the doses was effective in reducing the apoptotic cell counts and enhanced the memory and cognitive abilities. Rutin supplementation of-fered significant protection against anesthetic induced neurodegeneration and learning and memory disturbances.

Keywords: Neuroapoptosis, propofol, rutin, sevoflurane

Introduction

Every year millions of children and infants undergo surgery as a part of health care. Pediatric surgeries require the administration of general anesthetics. Mounting reports have demonstrated that general anesthetics induce intense neuroapoptosis in the developing brain, and can cause long-term cognitive deficits [1-4]. The developing brain has high plasticity and the exposures to general anesthetics in young children under the age of 4 may affect learning disabilities, including reading, lan-guage and math [5].

However mechanism’s underlying neuronal apoptosis mediated by anesthetics in the developing brain is still under investigation. Many possible mechanisms have been pro-posed including, activation of gamma-amino-butyric acid (GABA) receptors and inhibition of

N-methyl-D-aspartate (NMDA) receptors and associated impairment of synaptogenesis [1, 6-8], disruption of intracellular calcium homeo-stasis [9-11], activation of P75 neurotrophin receptors [12, 13] and regulation of cell cycle [14].

Sevoflurane, one of the most frequently used volatile anesthetics is especially useful for infants and children because of its properties of rapid induction and recovery together with less irritation to the airway [15, 16]. Many stud-ies have shown neonatal exposure to sevoflu-rane causing learning disabilities and memory deficits [16-18]. General anesthetic, propofol, blocks NMDA receptors and potentiates GABAA receptors [19]. At clinically relevant concentra-tions and durations, propofol causes apoptosis in the developing brain [20, 21] and associated cognitive dysfunction as well [22, 23].

Effect of rutin on sevoflurane and propofol anesthesia

14398 Int J Clin Exp Med 2015;8(8):14397-14409

Previous studies suggest the immature brain is more vulnerable to anesthetic-induced neuro-toxicity during the period of rapid synaptogene-sis [1]. Activation of extrinsic and intrinsic apop-totic pathways are involved in the anesthetic-induced neuroapoptosis [24].

These observations have raised serious con-cerns regarding the safe use of general anes-thesia in pediatric medicine. Strategies that can possibly suppress the associated neuro-toxicity of the anesthetics call for more research. Recent researches are focussing on plant products as a means of therapy for vari-ous medical conditions. Bai et al. [25] reported that reseveratrol, a phytoalexin found in grapes and other berries was able to offer protection against isoflurane-induced neurotoxicity. Pte- rostilbene, a flavonoid was observed to signifi-cantly improve cognitive performance of mice [26, 27]. Rutin, a bioflavonoid compound, is a glycoside derivative of quercetin, a polyphenol widely found in citrus fruits and the rinds of grapes and lime, in berries, including cranber-ries [28] as well as in buckwheat and aspara-gus [29].

Various pharmacological properties of rutin including-anticancer [30], anti-inflammatory [31] and antioxidant [32] effects have been reported. Further, rutin may also have thera-peutic potential for the treatment of neurode-generative diseases associated with oxidative stress [33]. Qu et al. [34] reported potent thera-peutic effect of rutin in cognitive deficits.

Thus, considering the protective effects of rutin, the present work aimed to investigate its effect in modulating apoptosis and improving cognitive deficits in sevoflurane or propofol-induced anesthesia in neonatal rodent model.

Materials and methods

Animals

All experiments were carried out in accordance with approved institutional animal care guide-lines of the University Hospital. The C57BL/6 pregnant mice (Guangdong Medical Laboratory Animal Co., China) were used in this study and were maintained in 12 h light/dark cycle at room temperature (22° ± 1°C). Mice had ad libitum access to water and food. The animals were housed individually in separate cages and

monitored closely for the day of birth, which was noted as postnatal day 0 (P0). The pups (male and female) were kept in cages in a 12 h light/dark cycle with free access to water with their littermates. Separate groups of mice were administered rutin orally at 25 or 50 mg/kg b.wt from P2 to P21. On P7, the mice were fur-ther randomly assigned to different treatment groups. Control pups received no rutin or anes-thesia. Treatment pups received either sevoflu-rane and/or propofol on P7.

Chemicals and reagents

Sevoflurane and propofol were purchased from Sigma-Aldrich, St.Louis, MO, USA. Fluoro-Jade C (0.001%) was obtained from Merck Millipore, Billerica, MA, USA. All other chemicals used in the study were of analytical grade and were obtained from Sigma-Aldrich, (St.Louis, MO, USA) unless otherwise specified.

Anaesthesia exposure

On postnatal day 7 (P7), mice were placed in a humid chamber with manipulating gloves and exposed to anesthetics. The total gas flow was 2 L/min, using 25% O2 as a carrier. Oxygen and anesthetic agent fractions were measured using a gas analysis system (Capnomac Ultima, GE Healthcare, Tokyo, Japan). During exposure to the anesthetic, mice were kept warm on a mat heated to 38° ± 1°C. Neonatal mice were assigned to receive 2.9% sevoflurane for 6 h in 30% oxygen [35] or a single intraperitoneal (i.p) injection of propofol at 150 mg/kg b.wt [20, 36] or a combined dose of propofol and sevoflu-rane. In combined dose, propofol injection (150 mg/kg) was followed by exposure to 2.9% sevo-flurane for 6 h.

Determination of plasma S100β

Following anesthetic exposure, S100β levels in the blood of mice were determined using Sangtec 100 ELISA kit (DiaSorin Inc, Stillwater, MN, USA) as per manufacturer’s instructions and as previously described [37]. Briefly, blood from each mouse was drawn from the left ven-tricle and was centrifuged for separation of plasma 2 h after anesthesia exposure. Fifty μL plasma was placed in each well of microtiter plate and mixed with 150 μL tracer from kit, incubated for 2 h, followed by addition of 3,3’,5,5’ tetramethylbenzidine substrate and


14399 Int J Clin Exp Med 2015;8(8):14397-14409

stop solution. The absorbance was read at 450 nm and the concentration of S100β was mea-sured using a standard curve.

Evaluation of neuroapoptosis

Apoptosis was evaluated by immunohisto-chemical staining for activated caspase-3 and Fluoro-Jade C staining. Five hours following exposure to anesthesia experimental treat-ments, mice were perfused transcardially with 0.1 M phosphate buffer containing 4% parafor-maldehyde. Brain sections were prepared and processed for activated caspase-3 immunos-taining using a well-established procedure for measuring neonatal apoptosis in the develop-ing brain [7, 35]. Immunohistochemistry was performed as described previously [38]. Briefly, the brain tissue sections were paraffin-embed-ded (5 µm thick) and were incubated overnight with anti-cleaved caspase-3 primary antibody (1:200; monoclonal antibody, Cell Signaling Technology, Beverly, MA, USA) at 4°C, followed by incubation with secondary antibody (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for about 40 min. The sections were fur-ther incubated with avidin-biotinylated peroxi-dase complex (Vectostain ABC-Kit, Vector Lab, Burlingame, CA, USA) for 40 min. The brain tis-sue sections were then stained with diamino-benzidine (DAB, Vector Laboratories, Burlin- game, CA, USA). Caspase-3 positive cells in various sections of the brain tissue-hippocam-pal CA1, CA3 and dentate gyrus (DG) were ana-lyzed using NIS-Elements BR imaging process-ing and analysis software (Nikon Corporation, Japan). The density of cleaved caspase-3 posi-tive cells in various sections was calculated by dividing the number of caspase-3 positive cells by the area of the imaged brain region.

Further for analysis of apoptotic cell counts by Fluoro-Jade C stain, the tissues were sliced into 60 micron-thickness and every other slice was mounted and stained with Fluoro-Jade C, a marker very specific for neurodegeneration. The number of apoptotic cell counts was recorded as Fluoro-Jade positive cells using Nikon Eclipse 80i microscope under 20 × magnification.

Behavioral studies

Mice that were exposed to anesthetics on P7 were further subjected to behavioral tests such as open-field, elevated plus-maze, Y-maze, and

fear conditioning tests. The trails were per-formed as described by Satoh et al. [39]. The movement of each mouse was monitored and analyzed using a computer-operated video tracking system (ANY-maze video tracking sys-tem, Stoelting Co., Wood Dale, IL, USA). In tasks using an apparatus with arms, arm entry by the mouse was counted when all four legs of the animal entered each arm.

The responses of mice to a new environment were measured by an open-field test using P35 mice. The responses were recorded as the total distance travelled (meters) in 10 min. The ele-vated plus-maze test was used to evaluate anx-iety-related behaviour. P35 mice were used in the study. The elevated plus-maze consisted of two open arms (25 × 5 cm) and two enclosed arms, with all arms elevated to a height of 50 cm above the floor. The behaviour of mice was monitored during a 10 min test period. The per-centage of time the mice spend in the open arms was considered as an index of anxious- behaviour.

The Y-maze test assesses spatial working memory of mice following anesthesia exposure at P7. Symmetrical Y-maze consists of three arms (25 × 5 cm) separated by 120° with 15 cm high transparent walls. Each mouse (P35) was placed in the centre of the Y-maze and allowed to freely explore the maze for a time period of 8 min. The total number of arms entered by each mouse was recorded. The per-centage of alternations in the behaviour of mice was calculated as the number of triads containing entries into all three arms divided by the maximum possible number of alternations (total number of arm entries minus 2) × 100.

Fear conditioning test

This test evaluates the hippocampal-depen-dent and hippocampal-independent learning. The test was performed as previously described [16]. Briefly, the P35 mice were subjected to conditioning trial for contextual and cued fear conditioning that consisted of a 5 min explora-tion period followed by three conditioned stimu-lus-unconditioned stimulus pairings parted by 60 sec time for each. The unconditioned stimu-lus consisted of 1 mA foot shock with 1 sec duration and an 80 db white noise is the condi-tioned stimulus of 20 sec duration. Un- conditioned stimulus was delivered during the last seconds of conditioned stimulus. A contex-


14400 Int J Clin Exp Med 2015;8(8):14397-14409

tual test was performed in the conditioning chamber for 5 min in the absence of white noise 24 h after conditioning. A cued test (for the same set of mice) was performed by pre-senting a cue (80 db white noise, 3 min dura-tion) in an alternate context with distinct visual and tactile cues. The freezing response rate (absence of movement in any part of the body during first second) was scored automatically and used as a measure of fear memory.

Memory and learning studies-Morris water maze test

To assess memory and cognitive capabilities, mice that were exposed to anesthetics on P7 were subjected to spatial reference memory and learning assessments with the Morris water maze. The trials were performed as described previously by Li et al. [38]. All trials and swim paths were recorded with ANY-maze video tracking system (Stoelting Co., Wood Dale, IL, USA).

Escape latency

Mice were trained for 4 days (postnatal days 31-34) in the Morris water maze. A platform (10.3 cm diameter) was submerged in a circular pool (180 cm diameter, 50 cm depth) filled with warm water (23°C ± 2°C). Mice were trained in 2 sessions during a day. In each of the ses-sions, the mice were allowed to perform four trials in which they were released from one of the four randomly assigned release points. Each mouse was allotted to have two short and two medium swims per session. Animals were given a time of 60 sec to locate the hidden plat-form. If they failed to locate in 60 sec, they were guided to the platform. In either case, the mice were removed from the platform after 15 sec. Training sessions were conducted till the mice were able to locate the hidden platform in less than 15 sec (average time per session). The tri-als and swim paths were recorded with ANY-maze video tracking system that measures the time taken (latency) to find the platform (s), as well as other behavioural information obtained during the spatial reference memory test.

Cued trials

Cued trials were performed to determine any visual impairments and/or swimming difficul-ties. In this study, the pool was surrounded by a white cloth to hide the visual cues. In trial (4 trials per day), the mice were placed in a fixed

position of the swimming pool towards the wall and were allowed to swim to a randomly posi-tioned platform with a rod (cue) placed 20 cm above water level in any one of the quadrants of the pool. Sixty second was allotted to locate the platform and 30 sec to sit on the platform after which the mice were removed from the pool. If unable to locate a platform within 60 sec, the mice were gently guided. The time taken for each mouse to reach the cued platform and the swim speed was recorded.

Place trials

After the cued trials, the white curtains and cue rod were removed. The same mice were tested for place trials to determine the ability to learn the spatial relationship between distant cues and the submerged platform that was kept in the same place for all place trials. During place trials, mice were placed in a random position in the swimming pool, facing the wall, and time taken to reach the submerged platform posi-tioned in the pool was recorded.

Probe trials

Probe trials were conducted to evaluate memo-ry retention following 24 h after place trials. During the trial, submerged platform was removed and mice were placed in quadrant diagonally opposite from the previous platform location. Swimming time spent by each mice in the target quadrant (probe time) and the num-ber of times animal crossed the original posi-tion of the platform (platform-cross) were recorded.

Statistical analysis

All the values are represented as mean ± stan-dard deviation (SD). Values at P < 0.05 are con-sidered significant as determined by One-way Analysis of variance (ANOVA). The values were analyzed using SPSS software (version 17.0).

Results

Rutin reduces the intensive apoptotic neurode-generation due to neonatal anesthesia

Caspase-3 is the main cell death marker and executioner enzyme of the apoptotic cell death cascade [40, 41]. The most vulnerable brain region, the hippocampus, reveals neural degen-eration even on the exposure to the lowest sevoflurane concentration (1%) [18]. Caspase-3


14401 Int J Clin Exp Med 2015;8(8):14397-14409

positive cells were detected in the CA1, CA3 areas of hippocampus and in the dentate gyrus (DG) of the mice exposed to anesthetics. Sevoflurane at 2.9% and propofol at 150 mg caused intense apoptosis. The number of cas-pase-3 positive cell counts and Fluoro-Jade C positive cells were strikingly higher in the ani-mals that received combined dose of sevoflu-rane and propofol than in the animals that were exposed to either one of the anesthetics (Figure 1). Further the anesthetics induced more marked apoptosis in CA1 region than CA3 and DG irrespective of whether given as a single drug or combined with sevoflurane, exhibiting a

higher percentage of apoptotic counts than propofol. Rutin caused considerable reduction in the apoptotic cell counts at both the doses. Rutin at 50 mg was more effective in markedly reducing apoptosis positive cells in pups whether exposed to sevoflurane and/or propo-fol. Rutin exhibited more efficiency against pro-pofol exposure.

Plasma S100β levels in pups exposed to anes-thesia on P7

S100β has been demonstrated as a useful bio-marker for the detection of anesthetic-mediat-ed neurodegeneration [37, 42]. S100β, the β

Figure 1. Rutin reduces the intensive apoptotic neurodegeneration due to neonatal anesthesia. Values are repre-sented as mean ± SD, n = 6. *represents statistical significance at P < 0.05 compared against control as determined by one-way ANOVA.


14402 Int J Clin Exp Med 2015;8(8):14397-14409

isomer of S100, appears to be released into the extra-cellular space near the injured tissue and can enter into the serum from the brain through a disrupted blood brain barrier after even mild brain injury secondary to trauma, hypoxia, ischemia and neurotoxin, etc. [43]. Consistent with the apoptotic cell counts observed, anesthetic exposure caused a multi-fold raise in plasma S100β levels in the order sevoflurane + propofol > sevoflurane > propofol (Figure 2). Though plasma S100β were higher following propofol exposure, the raise was not much significant as compared to control pups that were not exposed to anesthesia. Nevertheless, rutin supplementation to neona-tal mice significantly (P < 0.05) reduced the lev-els of S100β with higher rutin dose exhibiting more efficiency. Rutin however showed more potent effects against propofol exposure than sevoflurane, as propofol > sevoflurane > propo-fol + sevoflurane. Rutin brought the levels of S100β to almost near to control levels.

Influence of rutin supplementation on the be-haviour of neonatal mice following sevoflurane and propofol exposure on P7

To examine behavioral activity of the mice treat-ed with sevoflurane and/or propofol on P7, an open-field test was performed on P35. There

were observable changes in the behaviour of the mice exposed to propofol and/or sevoflu-rane as compared against control mice not exposed to anesthesia (Figure 3A). Rutin administration caused negligible changes in the behaviour of mice. Similar results as in open field test were observed in elevated-maze test (Figure 3B). Sevoflurane showed alteration as compared to control mice, however no sig-nificant changes were found in mice induced with propofol alone. Combined exposure to sevoflurane and propofol caused pronounced alterations than sevoflurane or propofol given as a single drug. Rutin at both the doses (25 mg and 50 mg) was able to effectively prevent the behavioural changes induced by anesthesia.

Working memory could be said as the ability to hold information temporally to do complex cog-nitive tasks and it involves both the hippocam-pus and prefrontal cortex [44, 45]. In order to examine whether exposure of the developing brain to, sevoflurane and/or propofol was asso-ciated with changes in spatial working memory, the mice were tested in a Y-maze task. The experiment examines whether the mice were able to remember the position of the arm selected in the preceding choice. By nature, rodents normally look out for a new arm, differ-

Figure 2. Plasma S100β levels in P7 mice following anesthesia exposure. Values are represented as mean ± SD, n = 6. *represents statistical significance at P < 0.05 compared against control as determined by one-way ANOVA.


14403 Int J Clin Exp Med 2015;8(8):14397-14409

ent from that selected in the previous choice. If the work-ing memory is impaired, the number of correct choices would be reduced in the Y-maze task.

Mice exposed to anesthetics, propofol and/or sevoflurane exhibited altered performanc-es as against control mice. In the mice that were supple-mented with rutin, these alterations were not signifi-cant as compared to anesthe-sia exposure without rutin (Figure 3C). The disturbances were more noticeable in mice exposed to 2.9% sevoflu-rane + 150 mg propofol. The results indicate that anesthe-sia exposure had significantly impaired performance of the mice irrespective of whether given alone or as combined drugs. Rutin treatment at both the doses caused a marked improvement in the working memory of mice.

The P36 mice were examined in a contextual/cued fear con-ditioning test to assess mem-ory following conditioning. The freezing responses of mice exposed to sevoflurane and/or propofol were signifi-cantly reduced in the contex-tual test compared with those of controls (Figure 3D). The

Figure 3. Influence of rutin on the general behavior and spatial working memory of mice. Rutin administration improved the gen-eral behavior of mice in a novel environment (A) and in contex-tual and cued fear conditioning (D). Improved performances were observed in elevated maze t (B) and Y-maze tests (C). Values are represented as mean ± SD, n = 6. *represents statistical sig-nificance at P < 0.05 compared against control as determined by one-way ANOVA.


14404 Int J Clin Exp Med 2015;8(8):14397-14409

startled freezing responses showed by the mice that were supplemented with rutin were considerably higher than the percentage of responses expressed by the mice that were exposed to anesthesia but not rutin administered.

Influence of rutin supplementation on learning and memory

The mice exposed to anesthesia were subject-ed to Morris Water Maze (MWM) testing to eval-uate the effect of neonatal exposure with sevo-flurane and/or propofol on potential learning and memory deficits. MWM is a reliable mea-sure of hippocampus-dependent spatial navi-gation and reference memory [46].

The P35 mice that were exposed to anesthesia on P7 were trained to explore the swimming pool and to reach on the platform. The escape latency of the mice was recorded as the time taken to reach the platform. With the training sessions, escape latency of the mice was found to gradually decrease for all the mice irrespec-tive of whether exposed to anesthesia alone or were treated with rutin. However the mice exposed to anesthesia without rutin were found to take a longer time to reach the platform. Rutin significantly reduced the escape latency

in both sevoflurane and propofol treatment mice and in mice exposed to sevoflurane and propofol (Figure 4).

Cued trials were conducted on P35 to evaluate swimming and visual abilities. The mice that were exposed to anesthesia took a consider-ably (P < 0.05) longer time to reach the plat-form when compared to control pups that received no anesthesia. The duration was much longer in sevoflurane and propofol exposed mice as compared to mice that were exposed to either sevoflurane or propofol. The mice that received rutin at both the doses were able to reach the platform much quicker. However, mice that received higher dose of rutin reached the platform at a lesser time as against those which received lower dose (Figure 5).

Place and probe trials were conducted to evalu-ate the differences in visual judgments and memory. The trials assess the ability of mice to learn and remember the location of a new plat-form (Figure 5). Rutin supplementation to the neonatal mice showed a significant improve-ment in performance and the mice were able to reach the platform in a lesser time than the anesthesia alone treated mice. Rutin at both doses was observed to be more effective on

Figure 4. Escape latency of P35 mice following exposure to anesthesia on P7. Values are represented as mean ± SD, n = 6. *represents statistical significance at P < 0.05 compared against control as determined by one-way ANOVA.


14405 Int J Clin Exp Med 2015;8(8):14397-14409

propofol than on sevoflurane alone or in combi-nation with propofol. However, the differences were negligible in place trials.

As illustrated in Figure 5, in probe trials the mice that were exposed to propofol and/or sevoflurane tend to spend less percentage of time in the target quadrant than mice in the control group unexposed to anesthesia. There was a statistically significant difference between the groups (P < 0.05) of normal con-trol mice and anesthesia control mice. Mice that were exposed to sevoflurane and propofol exhibited more alterations in the swim speed and swim path than those treated with sevoflu-rane or propofol as single anesthetic. Combined anesthetics exhibited to have a higher impact on memory. Rutin at 25 mg or 50 mg recorded a higher probe time as-propofol + rutin > sevo-flurane + rutin > sevoflurane + propofol+ rutin. Thus the treatment with rutin at both the doses was found to have improved the memory and learning ability of mice.

Discussion

Exposure to general anesthetics has been demonstrated to cause apoptotic neurodegen-

eration in the developing brains and subse-quent cognitive dysfunctions [47-50]. Clinical retrospective studies have reported that anes-thesia and surgery in children increase the risk of developing cognitive disabilities [51, 52]. Research in animal models has demonstrated that volatile anesthetics including isoflurane and sevoflurane could cause neuronal death if exposed at early stages of postnatal brain development [16, 48, 53]. Many of these stud-ies reported long-term neurocognitive abnor-malities [35, 47, 48]. These observations lead to further research in the use of anesthetics in pediatric surgeries. The present study evalu-ates the effectiveness of rutin in neonatal mice exposed to sevoflurane and propofol anaesthesia.

Cell death due to apoptosis is a vital part of nor-mal brain maturation, removing about 50-70% of neurons and progenitor cells [55, 56]. However, during brain development, neuro-apoptotis exceeding the natural apoptotic rate can be triggered by various pathologic process-es as hypoxia-ischemia, lack of neurotrophic factors, or due to prolonged exposure to anes-thetics [57, 58].

Figure 5. Learning and memory of mice following anesthesia exposure on P7 as determined by cued, place and probe trials with Morris Water maze. Values are represented as mean ± SD, n = 6. *represents statistical signifi-cance at P < 0.05 compared against control as determined by one-way ANOVA.


14406 Int J Clin Exp Med 2015;8(8):14397-14409

Neuroapoptosis following exposure to sevoflu-rane and/or propofol presented significant increase in caspase-3 positive cells. Caspase-3, a member of the caspase family, plays a central role in execution of apoptosis cascade and is well accepted as a biomarker for cell death by apoptosis [36, 59]. Previous studies indicate that sevoflurane [16, 53] and propofol [36, 58] could cause neurodegeneration in the develop-ing brains of neonatal rodent models. In our investigation, rutin (25 mg and 50 mg) effec-tively lowered caspase-3 positive cell counts in the hippocampal CA1 and CA3 and in DG regions of the brain. Although anesthetic-induced neurodegeneration has been found in many brain regions, our study focused on hip-pocampus, as previous reports have demon-strated that neonatal rats show normal short-term memory, a function predominantly involv-ing the prefrontal cortex with severe hippocam-pal lesion [60]. Robust neurogenesis ensures hippocampal learning [61], whereas decreased neurogenesis impairs it [62, 63].

The levels of neuropaoptosis correlated with the levels of plasma S100β, a neurodegenera-tive biomarker in blood. Previous studies have shown similar elevations following anesthesia [37, 42]. Marked decreases in apoptotic cell counts and plasma S100β in rutin administra-tion suggest that rutin was able to efficiently protect the neurons against anesthetic insult.

In addition to neuroapoptosis, sevoflurane and/or propofol administration produced neurocog-nitive deficits in mice at 5 weeks of age. Long-term memory and working memory were impaired. MWM test was used to evaluate long-term spatial learning/memory that involves a sequence of specific molecular processes in the hippocampal CA1 region. The results sug-gest impaired working memory and learning. It is widely recognized that the effects of anes-thetics on subsequent spatial learning/memo-ry are associated at least partially with damage to the hippocampal region [58]. Earlier reports also demonstrated that sevoflurane and as well as propofol exposure can induce neuronal apoptosis and also decrease cognition in mice [17, 58, 64].

Working memory refers to cognitive functions that provide concurrent temporary storage and manipulation of the informations that are vital and are required to perform complex cognitive

tasks [65]. Working memory is involved in high-er cognitive functioning as planning and sequential execution of tasks.

Neurogenesis in the brain proceeds throughout adulthood and impaired adult neurogenesis has been suggested to be associated with defi-cits in hippocampal-dependent memory includ-ing working memory [66-68]. In this study, it is notable that sevoflurane and propofol induced neuroapoptosis could be possibly attributed to be responsible for learning and memory defi-cits. Anesthetic exposure also affected the gen-eral behaviour of mice in open field tests, ele-vated and Y-maze tests. The observed improve-ments in the memory and behaviour of mice may possibly be due to the reduction in neuro-apoptosis as observed in rutin administration. The exact mechanisms through which rutin offers neuroprotection and improves cognition and memory have to be unravelled, however, possible means could be by interfering with the caspase cascade.

Conclusion

The current study suggests that combination of sevoflurane and propofol drugs presented high-er neurotoxicity than sevoflurane or propofol when administered alone. Rutin exhibited potential neuroprotective effects against the anesthetics in the order-propofol > sevoflurane > sevoflurane + propofol. Rutin could be further investigated for the molecular events involved in neuroprotection.

Disclosure of conflict of interest

None.

Address correspondence to: Rui-Gang Zhou, Department of Children’s Nervous and Rehabili- tation, Jining No. 1 People’s Hospital, No. 6, Jiankang Road, Jining 272111, Shandong, China. Tel: 0086-537-2293366; Fax: 0086-537-2293366; E-mail: [email protected]

References

[1] Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early exposure to common an-esthetic agents causes widespread neurode-generation in the developing rat brain and per-sistent learning deficits. J Neurosci 2003; 23: 876-882.

mailto:[email protected]


14407 Int J Clin Exp Med 2015;8(8):14397-14409

[2] Ma D, Williamson P, Januszewski A, Nogaro MC, Hossain M, Ong LP, Shu Y, Franks NP, Maze M. Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain. Anesthesiology 2007; 106: 746-753.

[3] Stratmann G. Neurotoxicity of anesthetic drugs in the developing brain. Anesth Analg 2011; 113: 1170-1179.

[4] Shen X, Dong Y, Xu Z, Wang H, Miao C, Soriano SG, Sun D, Baxter MG, Zhang Y, Xie Z. Selective anesthesia induced neuroinflammation in de-veloping mouse brain and cognitive impair-ment. Anesthesiology 2013; 118: 502-515.

[5] Flick RP, Katusic SK, Colligan RC, Wilder RT, Voigt RG, Olson MD, Sprung J, Weaver AL, Schroeder DR, Warner DO. Cognitive and be-havioral outcomes after early exposure to an-esthesia and surgery. Pediatrics 2011; 128: e1053-1061.

[6] Zhao YL, Xiang Q, Shi QY, Li SY, Tan L, Wang JT, Jin XG, Luo AL. GABAergic excitotoxicity injury of the immature hippocampal pyramidal neu-rons’ exposure to isoflurane. Anesth Analg 2011; 113: 1152-1160.

[7] Brambrink AM, Evers AS, Avidan MS, Farber NB, Smith DJ, Martin LD, Dissen GA, Creeley CE, Olney JW. Ketamine-induced neuroapopto-sis in the fetal and neonatal rhesus macaque brain. Anesthesiology 2012; 116: 372-384.

[8] Istaphanous GK, Ward CG, Nan X, Hughes EA, Mccann JC, McAuliffe JJ, Danzer SC, Loepke AW. Characterization and quantification of iso-flurane-induced developmental apoptotic cell death in mouse cerebral cortex. Anesth Analg 2013; 116: 845-854.

[9] Wei HF, Liang G, Yang H, Wang QJ, Hawkins B, Madesh M, Wang S, Eckenhoff RG. The com-mon inhalational anesthetic isoflurane induc-es apoptosis via activation of inositol 1, 4, 5-trisphosphate receptors. Anesthesiology 2008; 108: 251-260.

[10] Lunardi N, Ori C, Erisir A, Jevtovic-Todorovic V. General anesthesia causes long-lasting distur-bances in the ultrastructural properties of de-veloping synapses in young rats. Neurotox Res 2010; 17: 179-188.

[11] Zhao X, Yang Z, Liang G, Wu Z, Peng Y, Joseph DJ, Inan S, Wei H. Dual effects of isoflurane on proliferation, differentiation and survival in hu-man neuroprogenitor cells. Anesthesiology 2013; 118: 537-549.

[12] Head BP, Patel HH, Niesman IR, Drummond JC, Roth DM, Patel PM. Inhibition of p75 neuro-trophin receptor attenuates isoflurane-mediat-ed neuronal apoptosis in the neonatal central nervous system. Anesthesiology 2009; 110: 813-825.

[13] Pearn ML, Hu Y, Niesman IR, Patel HH, Drummond JC, Roth DM, Akassoglou K, Patel PM, Head BP. Propofol neurotoxicity is mediat-

ed by p75 neurotrophin receptor activation. Anesthesiology 2012; 116: 352-361.

[14] Soriano SG, Liu Q, Li J, Liu JR, Han XH, Kanter JL, Bajic D, Ibla JC. Ketamine activates cell cy-cle signaling and apoptosis in the neonatal rat brain. Anesthesiology 2010; 112: 1155-1163.

[15] Lerman J, Sikich N, Kleinman S, Yentis S. The pharmacology of sevoflurane in infants and children. Anesthesiology 1994; 80: 814-824.

[16] Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, Ito M, Imaki J. Neonatal exposure to sevoflurane induces abnormal social behav-iours and deficits in fear conditioning in mice. Anesthesiology 2009; 110: 628-637.

[17] Bercker S, Bert B, Bittigau P, Felderhoff-Muser U, Buhrer C, Ikonomidou C, Weise M, Kaisers UX, Kerner T. Neurodegeneration in newborn rats following propofol and sevoflurane anes-thesia. Neurotox Res 2009; 16: 140-147.

[18] Zheng SQ, An LX, Cheng X, Wang YJ. Sevoflurane causes neuronal apoptosis and adaptability changes of neonatal rats. Acta Anaesthesiol Scand 2013; 57: 1167-1174.

[19] Irifune M, Takarada T, Shimizu Y, Endo C, Katayama S, Dohi T, Kawahara M. Propofol-induced anesthesia in mice is mediated by γ-aminobutyric acid and excitatory amino acid receptors, Anesth Analg 2003; 97: 424-429.

[20] Cattano D, Young C, Straiko MM, Olney JW. Subanesthetic doses of propofol induce neuro-apoptosis in the infant mouse brain. Anesth Analg 2008; 106: 1712-1714.

[21] Tu S, Wang X, Yang F, Chen B, Wu S, He W, Yuan X, Zhang H, Chen P, Wei G. Propofol induces neuronal apoptosis in infant rat brain under hypoxic conditions. Brain Res Bull 2011; 86: 29-35.

[22] Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikeanian K, Tenkova TI, Stefovska V, Turski L, Olney JW. Blockade of NMDA recep-tors and apoptotic neurodegeneration in the developing brain. Science 1999; 283: 70-74.

[23] Fredriksson A, Ponten E, Gordh T, Eriksson P. Neonatal exposure to a combination of N-methyl-d-aspartate and γ-aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 2007; 107: 427-436.

[24] Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V. Anesthesia induces neuronal cell death in the developing rat brain via the intrin-sic and extrinsic apoptotic pathways. Neuroscience 2005; 135: 815-827.

[25] Bai T, Dong DS, Pei L. Resveratrol mitigates isoflurane-induced neuroapoptosis by inhibit-ing the activation of the Akt-regulated mito-chondrial apoptotic signaling pathway. Int J Mol Med 2013; 32: 819-826.


14408 Int J Clin Exp Med 2015;8(8):14397-14409

[26] Joseph JA, Fisher DR, Cheng V, Rimando AM, Shukitt-Hale B. Cellular and behavioral effects of stilbene resveratrol analogues: implications for reducing the deleterious effects of aging. J Agric Food Chem 2008; 56: 10544-10551.

[27] Chang J, Rimando A, Pallas M, Camins A, Porquet D, Reeves J, Shukitt-Hale B, Smith MA, Joseph JA, Casadesus G. Low-dose pterostil-bene, but not resveratrol, is a potent neuro-modulator in aging and Alzheimer’s disease. Neurobiol Aging 2012; 33: 2062-20671.

[28] Ramassamy C. Emerging role of polyphenolic compounds in the treatment of neurodegen-erative diseases: a review of their intracellular targets. Eur J Pharmacol 2006; 545: 51-64.

[29] Suzuki T, Honda Y and Mukasa Y. Effects of UV-B radiation, cold and desiccation stress on rutin concentration and rutin glucosidase ac-tivity in tartary buckwheat (Fagopyrum tatari-cum) leaves. Plant Sci 2005; 168: 1303-1307.

[30] Nothlings U, Murphy SP, Wilkens LR, Henderson BE and Kolonel LN. Flavonols and pancreatic cancer risk: the multiethnic cohort study. Am J Epidemiol 2007; 166: 924-931.

[31] Stewart LK, Soileau JL, Ribnicky D, Wang ZQ, Raskin I, Poulev A, Majewski M, Cefalu WT and Gettys TW. Quercetin transiently increases en-ergy expenditure but persistently decreases circulating markers of inflammation in C57BL/6J mice fed a high-fat diet. Metabolism 2008; 57: S39-46.

[32] Khan MM, Ahmad A, Ishrat T, Khuwaja G, Srivastawa P, Khan MB, Raza SS, Javed H, Vaibhav K, Khan A, Islam F. Rutin protects the neural damage induced by transient focal isch-emia in rats. Brain Res 2009; 1292: 123-135.

[33] Park SE, Sapkota K, Choi JH, Kim MK, Kim YH, Kim KM, Kim KJ, Oh HN, Kim SJ, Kim S. Rutin from dendropanax morbifera leveille protects human dopaminergic cells against rotenone induced cell injury through inhibiting JNK and p38 MAPK signaling. Neurochem Res 2014; 39: 707-718.

[34] Qu J, Zhou Q, Du Y, Zhang W, Bai M, Zhang Z, Xi Y, Li Z, Miao J. Rutin protects against cognitive deficits and brain damage in rats with chronic cerebral hypoperfusion. Br J Pharmacol 2014; 171: 3702-3715.

[35] Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, McAuliffe JJ, Danzer SC, Loepke AW. Comparison of the neuroapoptotic proper-ties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neo-natal mice. Anesthesiology 2011; 114: 578-587.

[36] Yang B, Liang G, Khojasteh S, Wu Z, Yang W, Joseph D, Wei H. Comparison of Neurode- generation and Cognitive Impairment in

Neonatal Mice Exposed to Propofol or Isoflurane. PLoS One 2014; 9: e99171.

[37] Wang S, Peretich K, Zhao Y, Liang G, Meng Q, Wei H. Anesthesia induced neurodegeneration in fetal rat brains. Pediatr Res 2009; 66: 435-440.

[38] Li Y, Liang G, Wang S, Meng Q, Wang Q, Wei H. Effect of fetal exposure to isoflurane on post-natal memory and learning in rats. Neuropharmacology 2007; 53: 942-950.

[39] Satoh Y, Endo S, Ikeda T, Yamada K, Ito M, Kuroki M, Hiramoto T, Imamura O, Kobayashi Y, Watanabe Y, Itohara S, Takishima K. Extra- cellular signal-regulated kinase 2 (ERK2) knock down mice show deficits in long-term memory; ERK2 has a specific function in learn-ing and memory. J Neurosci 2007; 27: 10765-10776.

[40] Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312-1316.

[41] Zimmermann KC, Green DR. How cells die: apoptosis pathways. J Allergy Clin Immnol 2001; 108: S99-103.

[42] Liang G, Ward C, Peng J, Zhao Y, Huang B, Wei H. Isoflurane causes greater neurodegenera-tion than an equivalent exposure of sevoflu-rane in the developing brain of neonatal mice. Anesthesiology 2010; 112: 1325-1334.

[43] Bloomfield SM, McKinney J, Smith L, Brisman J. Reliability of S100B in predicting severity of central nervous system injury. Neurocritical Care 2007; 6: 121-138.

[44] Jones MW. A comparative review of rodent pre-frontal cortex and working memory. Curr Mol Med 2002; 2: 639-647.

[45] Saxe MD, Battaglia F, Wang JW, Malleret G, David DJ, Monckton JE, Garcia AD, Sofroniew MV, Kandel ER, Santarelli L, Hen R, Drew MR. Ablation of hippocampal neurogenesis impairs contextual fear conditioning and synaptic plas-ticity in the dentate gyrus. Proc Natl Acad Sci U S A 2006; 103: 17501-17506.

[46] D’Hooge R, De Deyn PP. Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev 2001; 36: 60-90.

[47] Brambrink AM, Evers AS, Avidan MS, Farber NB, Smith DJ, Zhang X, Dissen GA, Creeley CE, Olney JW. Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology 2010; 112: 834-841.

[48] Kodama M, Satoh Y, Otsubo Y, Araki Y, Yonamine R, Masui K, Kazama T. Neonatal desflurane exposure induces more robust neu-roapoptosis than do isoflurane and sevoflu-rane and impairs working memory. Anesth- esiology 2011; 115: 979-991.

[49] Paule MG, Li M, Allen RR, Liu F, Zou X, Hotchkiss C, Hanig JP, Patterson TA, Slikker WJ, Wang C.


14409 Int J Clin Exp Med 2015;8(8):14397-14409

Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys. Neurotoxicol Teratol 2011; 33: 220-230.

[50] Zhang Y, Xu Z, Wang H, Dong Y, Shi HN, Culley DJ, Crosby G, Marcantonio ER, Tanzi RE, Xie Z. Anesthetics isoflurane and desflurane differ-ently affect mitochondrial function, learning, and memory. Ann Neurol 2012; 71: 687-698.

[51] Kalkman CJ, Peelen L, Moons KG, Veenhuizen M, Bruens M, Sinnema G, de Jong TP. Behavior and development in children and age at the time of first anesthetic exposure. Anesth- esiology 2009; 110: 805-812.

[52] Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Gleich SJ, Schroeder DR, Weaver AL, Warner DO. Early exposure to anesthesia and learning disabili-ties in a population-based birth cohort. Anesthesiology 2009; 110: 796-804.

[53] Lu Y, Wu X, Dong Y, Xu Z, Zhang Y, Xie Z. Anesthetic sevoflurane causes neurotoxicity differently in neonatal nave and Alzheimer dis-ease transgenic mice. Anesthesiology 2010; 112: 1404-1416.

[54] Ramage TM, Chang FL, Shih J, Alvi RS, Quitoriano GR, Rau V, Barbour KC, Elphick SA, Kong CL, Tantoco NK, Ben-Tzur D, Kang H, McCreery MS, Huang P, Park A, Uy J, Rossi MJ, Zhao C, Di Geronimo RT, Stratmann G, Sall JW. Distinct long-term neurocognitive outcomes af-ter equipotent sevoflurane or isoflurane anaes-thesia in immature rats. Br J Anaesth 2013; 110: i39-46.

[55] Oppenheim RW. Cell death during develop-ment of the nervous system. Annu Rev Neurosci 1991; 14: 453-501.

[56] Rakic S, Zecevic N. Programmed cell death in the developing human telencephalon. Eur J Neurosci 2000; 12: 2721-2734.

[57] Loepke AW, Soriano SG. An assessment of the effects of general anesthetics on developing brain structure and neurocognitive function. Anesth Analg 2008; 106: 1681-1707.

[58] Yu D, Jiang Y, Gao J, Liu B, Chen P. Repeated exposure to propofol potentiates neuroapopto-sis and long-term behavioral deficits in neona-tal rats. Neurosci Lett 2013; 534: 41-46.

[59] Gown AM, Willingham MC. Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3. J Histochem Cytochem 2002; 50: 449-454.

[60] Sanders RD, Xu J, Shu Y, Januszewski A, Halder S, Fidalgo A, Sun P, Hossain M, Ma D, Maze M. Dexmedetomidine attenuates isoflurane-in-duced neurocognitive impairment in neonatal rats. Anesthesiology 2009; 110: 1077-1085.

[61] Kempermann G, Wiskott L, Gage FH. Functional significance of adult neurogenesis. Curr Opin Neurobiol 2004; 14: 186-191.

[62] Madsen TM, Kristjansen PE, Bolwig TG, Wortwein G. Arrested neuronal proliferation and impaired hippocampal function following fractionated brain irradiation in the adult rat. Neuroscience 2003; 119: 635-642.

[63] Rola R, Raber J, Rizk A, Otsuka S, VandenBerg SR, Morhardt DR, Fike JR. Radiation-induced impairment of hippocampal neurogenesis is associated with cognitive deficits in young mice. Exp Neurol 2004; 188: 316-330.

[64] Sanders RD, Sun P, Patel S, Li M, Maze M, Ma D. Dexmedetomidine provides cortical neuro-protection: impact on anaesthetic-induced neuroapoptosis in the rat developing brain. Acta Anaesthesiol Scand 2010; 54: 710-716.

[65] Baddeley A. Working memory. Science 1992; 255: 556-559.

[66] Snyder JS, Hong NS, McDonald RJ, Wojtowicz JM. A role for adult neurogenesis in spatial long-term memory. Neuroscience 2005; 130: 843-852.

[67] Winocur G, Wojtowicz JM, Sekeres M, Snyder JS, Wang S. Inhibition of neurogenesis inter-feres with hippocampus-dependent memory function. Hippocampus 2006; 16: 296-304.

[68] Denis-Donini S, Dellarole A, Crociara P, Francese MT, Bortolotto V, Quadrato G, Canonico PL, Orsetti M, Ghi P, Memo M, Bonini SA, Ferrari-Toninelli G, Grilli M. Impaired adult neurogenesis associated with short-term memory defects in NF-kappaB p50-deficient mice. J Neurosci 2008; 28: 3911-3919.

Documents

Original Article Efficacy of rutin in inhibiting neuronal apoptosis … · 2018. 8. 31. · Abstract: Sevoflurane and propofol are widely used in pediatric anesthesia. Neurotoxicity