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Effect of Minocycline on Behavioral-Cognitive Impairments Induced by Global Cerebral Ischemia
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Mohammad Khajenouri1    , Masoud Fereidoni *2 |
1- M.Sc in Animal Physiology, Department of Biology, Ferdowsi University of Mashhad, Mashhad, Iran.
2- Professor of Physiology (Neuroscience), Department of Biology, Ferdowsi University of Mashhad, Mashhad, Iran. , fereidoni@um.ac.ir |
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Keywords: Minocycline [MeSH], Reversal Learning [MeSH], Morris Water Maze Test [MeSH], Memory, Short-Term [MeSH], Anxiety [MeSH]
Article ID: Vol27-32 |
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Type of Study: Original Articles |
Subject:
Physiology - Pharmacology
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Abstract: (46 Views) |
Extended Abstract
Introduction
Stroke is a relatively prevalent condition, serving as the primary cause of adult disability and the fifth leading cause of mortality in the United States.
In the central nervous system and the retina, glutamate-which plays a critical role in cell death via excessive release-is secreted in high concentrations during ischemia/reperfusion. The occurrence of ischemia/reperfusion in brain tissue leads to an elevation in intracellular calcium concentration, ischemic depolarization, and glutamate efflux. These events exacerbate damage to adjacent regions and increase the production of reactive oxygen species (ROS). This, in turn, induces chemotaxis of other immune cells to the tissue, resulting in further ROS release and the secretion of pro-inflammatory factors. Furthermore, it promotes the polarization of M2 microglia, the disruption of the blood-brain barrier (BBB) in the affected area, and the activation of the p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. It also triggers death receptors, which ultimately culminate in the induction of apoptosis. Moreover, it causes the release of cytochrome C from the affected cells during the reperfusion phase, leading to the induction of cell death, and activates matrix metalloproteinases (MMPs), impairing the tissue's ability to regenerate. Global cerebral ischemia is characterized by a reduction or complete cessation of blood flow to the entire brain, which rapidly induces neuronal damage, particularly in the CA1 region of the hippocampus; the severity of this injury escalates as the duration of the ischemic event increases.
Minocycline is extensively distributed throughout the body. It crosses the BBB to a significant extent and is also secreted into the saliva. The concentration of minocycline in the cerebrospinal fluid (CSF) can reach levels as high as 35% of its serum concentration. This study was conducted to evaluate the effect of minocycline on behavioral-cognitive impairments induced by global cerebral ischemia.
Methods
In this experimental study, 56 male Wistar rats (weighing 220–280 g) were randomly assigned to the following groups:
Control group: This group consisted of healthy animals that received no intervention.
Solvent group: Animals in this group comprised healthy animals that received a specific daily volume of the drug solvent (physiological saline), according to body weight and under conditions identical to the drug treatment groups.
Surgery group: Animals in this group underwent the same surgical procedures, in which the duration of anesthesia and exposure of the surgical site was similar to that of the ischemia groups; however, no ischemia was induced (with no carotid artery occlusion).
Ischemia group (stroke induction without receiving drug and solvent): Cerebral ischemia was induced through carotid artery occlusion for 20 minutes using microvascular clips. Animals in this group received no drug or solvent injections.
Ischemia + Saline group (stroke induction with daily solvent injection): Cerebral ischemia was induced through carotid artery occlusion for 20 minutes using microvascular clips. Animals in this group received daily injections of physiological saline. The stroke induction was performed using the ischemia-reperfusion technique.
To induce ischemia-reperfusion, a longitudinal incision of approximately 1 cm was made in the skin of the animal’s anterior neck. The underlying muscles were retracted, and the common carotid arteries (CCAs) were carefully isolated from the surrounding muscles and the vagus nerve. Subsequently, both carotid arteries were occluded for 20 minutes using clips (ischemic period). After 20 minutes, the clips were removed to restore blood flow (reperfusion period).
Minocycline-treated stroke induction groups (minocycline at doses of 11.25, 22.50, and 45 mg/kg/bw): Cerebral ischemia was induced via common carotid artery occlusion for 20 minutes using microvascular clips. Animals in these groups received daily intraperitoneal injections of minocycline at dosages of 11.25, 22.50, and 45 mg/kg/bw, respectively.
Thirty minutes post-surgery, the drug or solvent was injected intraperitoneally. The intraperitoneal injections of the drug or solvent were continued daily at the same time as the initial postoperative dose for an additional six days (totaling seven days of treatment). On the seventh day following surgery, the open-field test was conducted first, followed by the Y-maze test. Subsequently, from day 8 to day 17, the Morris water maze (MWM) test was implemented to evaluate spatial long-term memory, as well as long-term memory following strategy switching and spatial learning.
Anxiety-like behavior and motor activity were assessed using the open-field test. Short-term memory function was evaluated through the Y-maze test by observing and measuring the animal's spontaneous alternation behavior. Additionally, spatial learning and memory were assessed using the MWM test (spatial learning and reversal spatial learning).
Results
In the behavioral assessment, no statistically significant difference was observed among the investigated groups regarding the total distance traveled. However, surgical intervention and stroke induction led to a significant increase in anxiety-like behaviors (P<0.001). Specifically, the ischemia and ischemia + saline groups spent significantly less time in the center of the apparatus compared to the control group (P<0.001). Conversely, the minocycline-treated group receiving minocycline at a dose of 45 mg/kg/bw exhibited a significant increase in the time spent in the center compared to the ischemia group (P<0.001).
According to the Y-maze test results, the induction of global cerebral ischemia significantly decreased the percentage of correct responses in the ischemia group compared to the control group (P<0.001). Conversely, treatment with minocycline at doses of 22.50 and 45 mg/kg/bw significantly increased the number of correct choices in the animals (P<0.05).
In the MWM test, during the first four days of the trial (from day 9 to day 12), the time spent swimming to reach the hidden platform gradually decreased compared to the first day (P<0.001). Conversely, the ischemia and ischemia + saline groups spent significantly more time locating the platform (P<0.001). In contrast, the administration of minocycline at various doses led to a significant reduction in time locating the platform, suggesting that the drug improved spatial learning performance.
Ischemia/reperfusion in the affected rats resulted in a significant decrease in the time spent swimming to reach the hidden platform compared to the ischemia group (at a dose of 45 mg/kg/bw) (P<0.001). Days 9 to 12 of the experimental procedure correspond to days 1 to 4 of the MWM test.
In the probe strial, ischemia/reperfusion-induced injury culminated in a significant reduction in the time spent in the target quadrant (Northwest [NW]) compared to the control group (P<0.001). Conversely, the administration of minocycline at doses of 22.50 and 45 mg/kg/bw led to a significant increase in the mean time spent in the target quadrant (P<0.001).
Following the probe trial and the reversal learning phase, the mean time duration to locate the platform in the ischemia and ischemia + saline groups was significantly higher than that of the control group on days 6, 7, and 8 (P<0.001). During the reversal learning phase of the probe trial, the mean time spent to locate the platform in the groups receiving minocycline at doses of 11.25, 22.50, and 45 mg/kg/bw resulted in a significant reduction in the swimming time required to reach the hidden platform compared to the ischemia group (P<0.001). Subsequently, on day 9 of the MWM test-which corresponded to day 17 of the protocol-the reverse probe trial (or probe 2) was conducted. The injury induced by the ischemia/reperfusion process resulted in a significant reduction in the time spent in the target quadrant (Southwest [SW]) compared to the control group (P<0.001). This impairment in spatial memory performance was accompanied by alterations in learning strategies and the retrieval process. Conversely, treatment with minocycline at doses of 22.50 and 45 mg/kg/bw in the ischemia group led to a significant increase in the mean time spent in the target quadrant (P<0.001).
Furthermore, a comparison of the distance traveled on the first day revealed no significant differences between groups, indicating that motor activities were not affected by the experimental conditions.
Conclusion
Based on the results of this study, the administration of minocycline significantly improved cognitive function, memory, and anxiety levels without impairing motor activities following cerebral ischemia, particularly at dosages of 22.50 and 45 mg/kg. The observed improvements at these specific doses are likely attributable to the drug's neuroprotective capabilities and its efficacy in preventing cell death. These therapeutic effects were evident at the 22.50 and 45 mg/kg doses, whereas the 11.25 mg/kg dose proved insufficient to elicit a significant response. In other words, the drug's restorative effects demonstrated a dose-dependent pattern.
Ethical Statement
This study was approved by the Research Ethics Committee at Ferdowsi University of Mashhad (IR.UM.REC.1401.265).
A preliminary abstract of this research (excluding the MWM results) has been presented as a poster at the 12th Basic and Clinical Neuroscience Congress.
Funding
This article has been derived from the Master’s thesis of Mohammad Khajenouri (approval code: 59524/3) in Biology, specializing in Animal Physiology, at the Faculty of Science, Ferdowsi University of Mashhad.
Authors' Contributions
Mohammad Khajenouri (M.Sc): Project execution, Data collection, Data analysis, Interpretation of the results, Drafting of the initial manuscript.
Masoud Fereidoni (Ph.D): Project administration and design, Project execution, Data collection, Data analysis, Interpretation of the results, Approval of the final manuscript.
Conflicts of Interest
No conflicts of interest.
Acknowledgement
The authors would like to thank Ferdowsi University of Mashhad for providing the financial support for this study. We also wish to thank the staff of the Animal Physiology Research and Teaching Laboratory (especially Ms. Haghpeyma and
Ms. Khoshnoudi) in the Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, for their valuable assistance.
Key Message: Minocycline, particularly at doses of 22.50 and 45 mg/kg, improved cognitive function, memory, and anxiety-like behaviors in male Wistar rats without inducing motor impairment following cerebral ischemia.
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