Volume 32, Issue 1 (3-2025)
RJMS 2025, 32(1): 1-12 |
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Research code: 1400-1-233-52935
Ethics code: IR.TUMS.NI.REC.1400.015
Clinical trials code: A-10-7192-1rt
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Fahanik babaei J, Jalal Kamali S, Lajevardi N S, Rajabi M, Pestehei S K. Investigation of Repeated Exposure Effects to Ketamine During Preadolescence on Neurobehavioral Disorder and Oxidative Stress Outcomes in Adult Male and Female Wistar Rats. RJMS 2025; 32 (1) :1-12
URL: http://rjms.iums.ac.ir/article-1-7856-en.html
URL: http://rjms.iums.ac.ir/article-1-7856-en.html
Javad Fahanik babaei1 
, Sahra Jalal Kamali1 , Negin Sadat Lajevardi1 , Majid Rajabi2 , Seyed Khalil Pestehei *3
, Sahra Jalal Kamali1 , Negin Sadat Lajevardi1 , Majid Rajabi2 , Seyed Khalil Pestehei *3
1- Electrophysiology Research Center, Neuroscience Institute, Tehran University of Medical Sciences. Tehran, Iran
2- 0000-0001-9511-4382
3- Department of Anesthesiology, Tehran University of Medical Sciences, Tehran, Iran, & Neuroscience Institute, Tehran University of Medical Sciences. Tehran, Iran ,khalilpesteh@yahoo.com
2- 0000-0001-9511-4382
3- Department of Anesthesiology, Tehran University of Medical Sciences, Tehran, Iran, & Neuroscience Institute, Tehran University of Medical Sciences. Tehran, Iran ,
Abstract: (812 Views)
Background & Aims: Repeated exposure to general anesthesia during childhood and preadolescence is often unavoidable in a variety of medical procedures, including repeated radiotherapy sessions for pediatric cancers, treatment of severe burns, and laser-based interventions for congenital skin disorders. Growing evidence from both clinical and experimental studies suggests that exposure to anesthetic agents during critical periods of brain development may interfere with normal neurodevelopmental processes and contribute to long-lasting cognitive, behavioral, and neurochemical abnormalities. Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, is one of the most widely used anesthetic and analgesic agents in pediatric medicine due to its rapid onset, cardiovascular stability, and safety profile. However, increasing concerns have emerged regarding its potential neurotoxic effects on the developing brain. Previous animal and human studies have reported associations between early-life exposure to ketamine and impairments in learning, memory, social interactions, emotional regulation, and other neurobehavioral functions. Although several studies have examined the acute and short-term consequences of ketamine administration, limited information is available regarding the persistent effects of repeated ketamine exposure during the preadolescent period and the extent to which these alterations continue into adulthood. Therefore, the present study was designed to evaluate the long-term behavioral and biochemical consequences of repeated ketamine administration during preadolescence and to determine whether such exposure induces persistent neurobehavioral deficits and oxidative stress in adult male and female Wistar rats.
Methods: In this experimental study, thirty-two preadolescent Wistar rats of both sexes were selected randomly from four different litters. After weaning, animals at postnatal day 23, weighing approximately 35–40 g, were assigned to four experimental groups (n = 8 per group): male control, female control, ketamine-treated male, and ketamine-treated female groups. Animals in the ketamine groups received intraperitoneal injections of ketamine at a dose of 20 mg/kg once daily for 14 consecutive days, from postnatal day 25 to day 38. Control groups received standard handling and care under identical environmental conditions. Body weight and general behavioral conditions were monitored throughout the treatment period. Following the final ketamine injection, all animals were maintained under standard laboratory conditions for an additional 30–35 days to allow maturation into adulthood. Behavioral assessments were then conducted to investigate long-term cognitive and emotional outcomes. Spatial learning and memory performance were evaluated using the Morris Water Maze (MWM) test. During the acquisition phase, animals underwent four training trials per day for three consecutive days to locate a hidden platform submerged beneath the water surface. Memory retention was examined on the fourth day using a probe trial in which the platform was removed, and parameters including escape latency and time spent in the target quadrant were analyzed. Anxiety-like behavior and locomotor activity were assessed using the Open Field (OF) test. Animals were allowed to freely explore the arena for five minutes while behavioral activity was recorded using a digital tracking system. Total distance traveled, frequency of rearing behavior, and grooming activity were measured as indicators of locomotor function and anxiety-related responses. After completion of behavioral testing, animals were sacrificed and whole-brain tissues were collected for biochemical analyses. Brain homogenates were prepared and oxidative stress parameters were evaluated. Total protein concentrations were determined using the bicinchoninic acid (BCA) assay. Reduced glutathione (GSH) levels were measured using Ellman’s reagent, whereas superoxide dismutase (SOD) activity was quantified using a commercially available assay kit based on inhibition of superoxide-mediated formazan formation. Statistical analyses were performed using one-way and two-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests. A value of P < 0.05 was considered statistically significant.
Results: Repeated ketamine administration during preadolescence produced significant long-term behavioral alterations that remained detectable in adulthood. During the treatment period, ketamine-treated animals exhibited significant changes in body weight gain compared with their corresponding control groups. Male rats demonstrated significantly greater weight gain during days 10–15 of treatment, whereas female rats showed significant differences from days 5–15. No significant sex-related differences in body weight gain were observed between ketamine-treated males and females. Behavioral evaluation in the Open Field test revealed marked alterations in locomotor and anxiety-related behaviors. Ketamine-exposed animals displayed increased locomotor activity compared with controls, as indicated by a greater total distance traveled within the testing arena. This increase reached statistical significance particularly in female rats. Furthermore, significant differences were observed between ketamine-treated male and female animals, suggesting possible sex-dependent sensitivity to ketamine exposure. Anxiety-like behavior was also significantly elevated following repeated ketamine administration. Both male and female ketamine-treated groups exhibited a significant increase in rearing behavior compared with their respective control groups, indicating heightened anxiety and exploratory responses. The increase was particularly pronounced in female animals. In contrast, grooming behavior did not differ significantly among experimental groups. Assessment of spatial learning and memory using the Morris Water Maze demonstrated that repeated ketamine exposure induced significant cognitive impairment. Ketamine-treated animals showed poorer performance during memory testing, characterized by increased latency to locate the target area and reduced retention of spatial information compared with control animals. These findings indicate persistent deficits in hippocampus-dependent spatial memory following preadolescent ketamine exposure.
Biochemical analyses revealed evidence of oxidative stress in adult animals previously exposed to ketamine. A significant reduction in SOD activity was observed in both male and female ketamine-treated groups compared with controls, suggesting impaired antioxidant defense mechanisms and increased susceptibility to oxidative damage. Interestingly, although brain GSH levels tended to be lower in ketamine-treated animals, these reductions did not reach statistical significance. Sex-related differences in oxidative stress responses were also observed, indicating that male and female animals may exhibit distinct neurochemical adaptations following repeated ketamine exposure.
Conclusion: The findings of the present study demonstrate that repeated ketamine administration during the preadolescent period produces persistent neurobehavioral and biochemical alterations that remain evident in adulthood. Exposure to ketamine during a critical stage of brain development resulted in long-lasting impairments in spatial memory, increased anxiety-like behaviors, altered locomotor activity, and significant disruption of antioxidant defense systems, as reflected by decreased SOD activity. These observations support the hypothesis that repeated exposure to ketamine during early developmental stages may interfere with normal neural maturation and contribute to enduring changes in brain function. Given the widespread use of ketamine in pediatric anesthesia, these results raise important concerns regarding its potential long-term neurological consequences. The present findings provide additional insight into the mechanisms underlying ketamine-induced neurotoxicity and suggest that oxidative stress may play an important role in mediating its long-term effects. Further investigations focusing on molecular pathways, structural brain alterations, and potential protective interventions are warranted to improve the safety of anesthetic practices in children and adolescents and to minimize the risk of long-term neurodevelopmental complications.
Methods: In this experimental study, thirty-two preadolescent Wistar rats of both sexes were selected randomly from four different litters. After weaning, animals at postnatal day 23, weighing approximately 35–40 g, were assigned to four experimental groups (n = 8 per group): male control, female control, ketamine-treated male, and ketamine-treated female groups. Animals in the ketamine groups received intraperitoneal injections of ketamine at a dose of 20 mg/kg once daily for 14 consecutive days, from postnatal day 25 to day 38. Control groups received standard handling and care under identical environmental conditions. Body weight and general behavioral conditions were monitored throughout the treatment period. Following the final ketamine injection, all animals were maintained under standard laboratory conditions for an additional 30–35 days to allow maturation into adulthood. Behavioral assessments were then conducted to investigate long-term cognitive and emotional outcomes. Spatial learning and memory performance were evaluated using the Morris Water Maze (MWM) test. During the acquisition phase, animals underwent four training trials per day for three consecutive days to locate a hidden platform submerged beneath the water surface. Memory retention was examined on the fourth day using a probe trial in which the platform was removed, and parameters including escape latency and time spent in the target quadrant were analyzed. Anxiety-like behavior and locomotor activity were assessed using the Open Field (OF) test. Animals were allowed to freely explore the arena for five minutes while behavioral activity was recorded using a digital tracking system. Total distance traveled, frequency of rearing behavior, and grooming activity were measured as indicators of locomotor function and anxiety-related responses. After completion of behavioral testing, animals were sacrificed and whole-brain tissues were collected for biochemical analyses. Brain homogenates were prepared and oxidative stress parameters were evaluated. Total protein concentrations were determined using the bicinchoninic acid (BCA) assay. Reduced glutathione (GSH) levels were measured using Ellman’s reagent, whereas superoxide dismutase (SOD) activity was quantified using a commercially available assay kit based on inhibition of superoxide-mediated formazan formation. Statistical analyses were performed using one-way and two-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests. A value of P < 0.05 was considered statistically significant.
Results: Repeated ketamine administration during preadolescence produced significant long-term behavioral alterations that remained detectable in adulthood. During the treatment period, ketamine-treated animals exhibited significant changes in body weight gain compared with their corresponding control groups. Male rats demonstrated significantly greater weight gain during days 10–15 of treatment, whereas female rats showed significant differences from days 5–15. No significant sex-related differences in body weight gain were observed between ketamine-treated males and females. Behavioral evaluation in the Open Field test revealed marked alterations in locomotor and anxiety-related behaviors. Ketamine-exposed animals displayed increased locomotor activity compared with controls, as indicated by a greater total distance traveled within the testing arena. This increase reached statistical significance particularly in female rats. Furthermore, significant differences were observed between ketamine-treated male and female animals, suggesting possible sex-dependent sensitivity to ketamine exposure. Anxiety-like behavior was also significantly elevated following repeated ketamine administration. Both male and female ketamine-treated groups exhibited a significant increase in rearing behavior compared with their respective control groups, indicating heightened anxiety and exploratory responses. The increase was particularly pronounced in female animals. In contrast, grooming behavior did not differ significantly among experimental groups. Assessment of spatial learning and memory using the Morris Water Maze demonstrated that repeated ketamine exposure induced significant cognitive impairment. Ketamine-treated animals showed poorer performance during memory testing, characterized by increased latency to locate the target area and reduced retention of spatial information compared with control animals. These findings indicate persistent deficits in hippocampus-dependent spatial memory following preadolescent ketamine exposure.
Biochemical analyses revealed evidence of oxidative stress in adult animals previously exposed to ketamine. A significant reduction in SOD activity was observed in both male and female ketamine-treated groups compared with controls, suggesting impaired antioxidant defense mechanisms and increased susceptibility to oxidative damage. Interestingly, although brain GSH levels tended to be lower in ketamine-treated animals, these reductions did not reach statistical significance. Sex-related differences in oxidative stress responses were also observed, indicating that male and female animals may exhibit distinct neurochemical adaptations following repeated ketamine exposure.
Conclusion: The findings of the present study demonstrate that repeated ketamine administration during the preadolescent period produces persistent neurobehavioral and biochemical alterations that remain evident in adulthood. Exposure to ketamine during a critical stage of brain development resulted in long-lasting impairments in spatial memory, increased anxiety-like behaviors, altered locomotor activity, and significant disruption of antioxidant defense systems, as reflected by decreased SOD activity. These observations support the hypothesis that repeated exposure to ketamine during early developmental stages may interfere with normal neural maturation and contribute to enduring changes in brain function. Given the widespread use of ketamine in pediatric anesthesia, these results raise important concerns regarding its potential long-term neurological consequences. The present findings provide additional insight into the mechanisms underlying ketamine-induced neurotoxicity and suggest that oxidative stress may play an important role in mediating its long-term effects. Further investigations focusing on molecular pathways, structural brain alterations, and potential protective interventions are warranted to improve the safety of anesthetic practices in children and adolescents and to minimize the risk of long-term neurodevelopmental complications.
Type of Study: Research |
Subject:
Anesthesiology
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