Exploring the Impact of Preoperative Sleep Fragmentation on Cognitive Function in Mice: The Role of Microglial Activation and Iron Metabolism

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Introduction
Perioperative neurocognitive disorders (PND) represent a spectrum of cognitive impairments that can occur in the perioperative period, encompassing postoperative cognitive dysfunction (POCD), postoperative delirium (POD), and long-term cognitive decline [1].Tese disorders, previously considered distinct entities, are now recognized as interconnected conditions that can signifcantly impact patients' quality of life and long-term outcomes postsurgery [2].As we continue to grapple with the complex etiologies and risk factors of PND, it has become evident that these conditions are not merely a product of surgical stress or anesthesia, but rather result from a multifactorial cascade involving patient's preoperative cognitive status, genetic predisposition, comorbid conditions, surgical factors, and postoperative care practices [3].In addition to the direct impact on cognitive function, PND can lead to prolonged hospital stays, increased healthcare costs, and higher rates of morbidity and mortality [4].Moreover, it also contributes to a loss of independence, posing signifcant challenges to patients' personal lives and social functioning [5].Despite its widespread prevalence and impact, PND remains underrecognized and underdiagnosed in clinical practice.Our understanding of the underlying mechanisms is still limited, and further research is urgently needed to identify efective strategies for the prevention, early detection, and management of these disorders.Terefore, it is crucial to shed light on the complexities of PND in order to improve patient outcomes in the perioperative period.
Te sleep fragmentation, characterized by repeated awakenings and disruptions in the sleep cycle, has been increasingly recognized as a signifcant preoperative risk factor for perioperative neurocognitive disorders (PND) in older adults [6].Te intricate relationship between sleep, aging, and cognitive function forms a critical triad in understanding the pathophysiology of PND.As people age, changes in sleep architecture, coupled with the increased prevalence of sleep disorders, contribute to higher levels of sleep fragmentation [7].In the context of surgery, the role of sleep becomes even more pertinent.Mounting evidence suggests that preoperative sleep fragmentation may predispose older adults to PND, making it a crucial area for investigation.Te underlying mechanism is believed to involve the dysregulation of neuroimmunological and neuroendocrine systems that are vital for maintaining cognitive function [8].Despite this knowledge, the interplay between preoperative sleep fragmentation and PND in older adults is not fully elucidated and remains an active area of research.It is essential to explore this relationship further, as it may open avenues for preventative strategies and interventions that can improve sleep quality, thereby reducing the incidence of PND and enhancing postoperative recovery.In this context, the development and implementation of comprehensive sleep evaluations in the preoperative period could be of particular relevance.Not only would this approach help in identifying patients at risk for PND but could also provide an opportunity for targeted sleep interventions prior to surgery.
Ferroptosis, derived from the Latin word "ferrum" meaning iron, and the Greek term "ptosis" for falling, is a novel type of programmed cell death characterized by irondependent accumulation of lipid peroxides [9].First coined in 2012, ferroptosis represents a distinct form of cell death from other well-known types such as apoptosis, necrosis, and autophagy [10].Unlike other forms of cell death that often involve the collapse of cellular structure or function, ferroptosis is uniquely dependent on the intracellular balance of iron and lipid peroxidation [11].It is believed to occur when cells sufer from an iron overload, resulting in the generation of reactive oxygen species (ROS) and subsequent lipid peroxidation, which ultimately leads to cell death [12].
However, despite the mounting evidence suggesting a link between sleep fragmentation, microglial iron metabolism, and the development of PND, the mechanisms underpinning this relationship remain elusive.Our study aims to elucidate these mechanisms and explore the potential of targeting microglial iron metabolism and ferroptosis as a novel strategy for preventing sleep fragmentation-induced PND in older adults.By delving into this relatively unexplored territory, we hope to shed light on the intricate network of processes linking sleep, iron metabolism, and cognitive function, potentially paving the way for new therapeutic strategies to mitigate the risk of PND in the elderly population.

Methods
2.1.Animals and Grouping.Adult male C57BL/6J mice, aged between 8-10 weeks, were accommodated in plastic enclosures, with their circadian rhythm regulated by a 12-hour light/dark cycle.Unrestricted access to food and water was ensured throughout the experiment.Te surrounding environment was meticulously maintained at a consistent temperature and humidity for the entire duration of research studies.All experimental procedures involving animals were conducted strictly following the guidelines provided in the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.Furthermore, the study received formal approval from the Ethics Committee on Laboratory Animal Use at the Second Afliated Hospital of Nanchang University.In this study, we employed both adult and aged male C57BL/6J mice to investigate the impact of sleep deprivation and pharmaceutical intervention on tibial fracture healing.Te mice were stratifed into several groups based on age and treatment regimens.For the adult cohort, we established four experimental groups: the adult control (AC) group was maintained under standard conditions; the adult tibial fracture (AT) group underwent a tibial fracture operation with internal fxation; the adult sleep deprivation (AS) group experienced 48 hours of sleep deprivation using a modifed multiple platform water method before any procedure; and the adult tibial fracture with preoperative sleep deprivation (ATS) group was subjected to the same sleep deprivation protocol before the tibial fracture operation.Parallel to the adult cohort, the aged mice were divided into four corresponding groups: the control (C) group, the tibial fracture (T) group, the sleep deprivation (S) group, and the tibial fracture with preoperative sleep deprivation (TS) group, each undergoing analogous treatments as their adult counterparts.In a separate set of experiments, the aged mice were allocated into four additional groups to assess the efects of pharmacological agents on fracture healing under sleep-deprived conditions.Te TS group was sleep-deprived for 48 hours prior to tibial fracture surgery.Te sleep deprivation with minocycline (SM) group received a daily dose of minocycline (30 mg/kg) for 6 days before undergoing sleep deprivation and surgery.Te sleep deprivation with deferoxamine (SD) group was administered deferoxamine (100 mg/kg) daily for 6 days followed by sleep deprivation and surgery.Lastly, the sleep deprivation with minocycline and deferoxamine (SMD) group received both minocycline and deferoxamine at the respective dosages before the sleep deprivation and surgical procedure.

Tibial Fracture Fixation.
Under general anesthesia induced by a combination of ketamine (75 mg/kg) and xylazine (10 mg/kg), administered via intraperitoneal injection, 2 Journal of Clinical Pharmacy and Terapeutics a midline skin incision was made in the adult male C57BL/6J mice over the anterior aspect of the right hindlimb.Te soft tissue was carefully dissected to expose the tibia.A controlled fracture was created using a three-point bending apparatus.A 0.38 mm stainless steel fxation pin was inserted retrogradely into the medullary canal of the tibia, across the fracture site, ensuring a stable internal fxation.Following this, the skin incision was sutured with a 5-0 absorbable thread.Postoperatively, the mice were returned to their housing cage and monitored regularly for signs of pain or distress.Analgesics (buprenorphine: 0.05-0.1 mg/kg) were administered as needed for pain relief.All experimental procedures were performed in adherence to the ethical guidelines stated in the guide and received approval from the Institutional Animal Care and Use Committee.

Sleep Fragmentation.
Te sleep fragmentation model was established by the improved sleep deprivation method in a multiplatform water environment.In brief, 10 circular platforms (5 cm high and 2 cm in diameter) were placed in a transparent water tank (20 cm × 30 cm × 40 cm), and the distance between each two platforms was 5 cm.Te water tank was flled with water to 1 cm below the platform, and the mice were placed on the platform for continuous sleep deprivation for 72 hours (if the mice slept, they would wake up because their heads would touch water or fall into the water), and they were continuously irradiated by fuorescent lamps.Sleep deprivation was performed continuously for 12 hours (i.e., 9 : 00∼21 : 00) every day.

Morris Water Maze
Test.Following the internal fxation procedure, the mice were given a recovery period of one week before the commencement of behavioral testing.Te Morris water maze (MWM) test was conducted to assess spatial learning and memory.Te water maze apparatus consisted of a round pool, 120 cm in diameter and 50 cm deep, flled with water maintained at a temperature of 22 ± 1 °C.Te pool was divided into four equal quadrants and a hidden platform (10 cm in diameter) was placed in one of the quadrants, submerged 1 cm below the water surface.Visual cues were provided around the maze to assist spatial orientation.Training trials were conducted twice daily for fve consecutive days.Each mouse was placed in the water facing the wall of the pool at one of the four starting locations, which were varied randomly.Te mice were allowed a maximum of 60 seconds to fnd the hidden platform.If a mouse failed to fnd the platform within the given time, it was gently guided to it.Each mouse was allowed to stay on the platform for 15 seconds before being taken out of the pool.On the sixth day, a probe trial was performed by removing the platform.Each mouse was allowed to swim freely for 60 seconds, and the time spent in the target quadrant (where the platform was previously located) was recorded to assess memory retention.Te movements of the mice were tracked and recorded using a computerized video system.Te parameters analyzed included the latency to fnd the hidden platform during training trials and the time spent in the target quadrant during the probe trial.

Fear Conditioning Test.
After a recovery period of one week postsurgery, the fear conditioning test was initiated.Te fear conditioning setup consisted of a conditioning chamber, equipped with a metal grid foor connected to a shock scrambler.Te chamber was cleaned with 70% ethanol between each mouse to remove any scent cues.On the frst day, termed the "conditioning day," mice were placed in the chamber for a period of 180 seconds for habituation.After this habituation period, an auditory tone (2000 Hz, 85 dB) was presented for 30 seconds, which coterminated with a 2-second mild foot shock (0.5 mA).Tis tone-shock pairing was repeated twice with an interval of 60 seconds between the pairings.Mice were returned to their home cage 60 seconds after the last shock.On the second day, termed the "context test," mice were placed back into the same conditioning chamber (without the tone and shock) for 5 minutes to measure fear to the context.Freezing behavior, defned as a complete lack of movement except for respiration, was recorded as an index of fear memory.On the third day, termed the "tone test," the conditioning chamber was altered (altered context) to reduce contextual cues.Mice were placed in the altered chamber for 180 seconds, followed by the same tone used during the conditioning day for another 180 seconds, but without any foot shock.Freezing behavior was again recorded to evaluate the conditioned fear response to the tone.Te degree of freezing was measured by a video tracking system, and the percentage of time spent freezing was analyzed for each phase of the experiment.

Detection of the Infammatory Cytokines and Oxidative
Stress Markers.At designated time points postsurgery, mice were humanely euthanized.Brains were rapidly removed and the hippocampi were carefully dissected on ice.Te tissue samples were then homogenized in a suitable bufer to prepare tissue lysates.To measure the levels of infammatory cytokines, the homogenates were subjected to centrifugation at 10,000 rpm for 10 min at 4 °C.Te supernatants were collected and stored at −80 °C until further use.Te concentrations of TNF-α, IL-6, and IL-10 in the supernatants were determined using specifc enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer's instructions.Absorbance was measured on a microplate reader, and cytokine concentrations were calculated based on the standard curve.To evaluate the levels of oxidative stress markers, the activities of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in the hippocampal homogenates were measured using specifc commercial assay kits according to the manufacturer's instructions.SOD activity was determined based on its ability to inhibit the oxidation of a substrate, with the results expressed in units/mg protein.MDA content, an indicator of Journal of Clinical Pharmacy and Terapeutics lipid peroxidation, was assessed by measuring the absorbance of the color produced during the reaction with thiobarbituric acid at 532 nm, with results expressed in nmol/ mg protein.

Evaluation of Microglia Activation and Ferroptosis
Markers.After euthanasia, hippocampal tissues were collected from the mice postsurgery and homogenized in a suitable RIPA bufer to prepare tissue lysates.Te lysates were centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatants were collected for protein quantifcation and subsequent analysis.Te protein concentrations in the supernatants were determined using the Bradford method, which was used for normalization of subsequent assays.Te expressions of CD68 protein and ferroptosis-related proteins, HCP1, TfR, Fpn1, DMT1, and GPX4, were evaluated using Western blot analysis.In brief, equal amounts of protein were separated by SDS-PAGE and then transferred onto PVDF membranes.Te membranes were blocked and incubated with primary antibodies against HCP1, TfR, Fpn1, DMT1, and GPX4 overnight at 4 °C.After washing, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies.Te protein bands were visualized using an enhanced chemiluminescence detection system.

Statistical Analysis.
All data collected during the study were processed and analyzed using statistical software (such as SPSS and GraphPad Prism).Data are expressed as mean ± standard deviation (SD) unless otherwise specifed.Before applying statistical tests, the normality of data distribution was assessed using the Shapiro-Wilk test.For normally distributed data, the diferences between the two groups were evaluated using the Student's t-test.For multiple group comparisons, one-way ANOVA followed by Tukey's post hoc test was used.If the data were not normally distributed, nonparametric tests were applied.Diferences between the two groups were assessed using the Mann-Whitney U test.For multiple group comparisons, the Kruskal-Wallis test followed by Dunn's post hoc test was performed.For repeated measures data, such as the fear conditioning test, a two-way repeated measure ANOVA was employed, followed by Bonferroni post hoc tests.Te correlation between diferent variables was determined by the Pearson correlation coefcient for normally distributed data, or the Spearman correlation coefcient for nonnormally distributed data.Te results were considered statistically signifcant at a p value of less than 0.05.All statistical tests were two-tailed.

Te Impact of Sleep Fragmentation (SF) on Cognitive Function in Adult and Aged Mice Posttibial Fracture Surgery (TFS).
In the results of the study, we assessed the cognitive function of adult and aged C57BL/6J mice following sleep fragmentation (SF) and tibial fracture fxation (TFS) using the Morris water maze and fear conditioning assays.For the Morris water maze, adult mice exhibited a consistent escape latency across all groups, with the adult control (AC group) averaging 18-20 seconds and the tibial fracture with preoperative sleep deprivation (ATS group) showing a slight increase to 24-26 seconds.Te number of platform crossings remained similar across adult groups, with a slight decrease in the ATS group.In contrast, aged mice demonstrated a more pronounced efect of treatments on cognitive function.Notably, the aged tibial fracture (T group), sleep deprivation (S group), and tibial fracture with preoperative sleep deprivation (TS group) showed a signifcant increase in escape latency times during the Morris water maze (76-98 seconds) compared to the control (C group), with the TS group displaying the most substantial delay (P < 0.05).
Moreover, the number of platform crossings was signifcantly reduced in these groups, with the TS group showing the least number of crossings (P < 0.05).Te fear conditioning assay results mirrored these fndings.In adult mice, the percentage of postoperative condition-induced freezing time showed minimal diferences between groups.However, aged mice in the T, S, and TS groups demonstrated a marked reduction in freezing time compared to controls, with the TS group showing the most signifcant decrease (P < 0.05), indicating a decline in associative memory after SF and TFS (Figure 1).

Terapeutic Efcacy of Minocycline and Deferoxamine on Cognitive Function in Aged Mice following Sleep
Fragmentation and Tibial Fracture Surgery.In this part, we used aged mice to establish a model of TFS, and mice were applied SF for 24 h, 48 h, and 72 h, respectively.Ten, we used the microglial inhibitor, minocycline, and/or the iron chelator, deferoxamine (DFO).We analyzed the results of the Morris water maze and fear conditioning experiments at diferent time points presurgery and postsurgery.Our fndings indicate that minocycline and deferoxamine treatments improved cognitive outcomes in aged mice subjected to sleep deprivation and tibial fracture surgery.In the Morris water maze, the escape latency decreased, and the number of platform crossings increased in the minocycline (SM), deferoxamine (SD), and the combined treatment groups (SMD) compared to the sleep-deprived surgery group (TS).Te fear conditioning results supported these fndings, with treated groups showing higher freezing times, suggesting better memory retention.Te combined treatment (SMD) group exhibited the most signifcant improvements in both tests, highlighting the potential synergistic efects of minocycline and deferoxamine in mitigating cognitive defcits following surgical trauma and sleep deprivation in aged mice (Figure 2).

Efect of Minocycline and Deferoxamine on Infammatory
Markers in Aged Mice Subjected to Sleep Deprivation and Surgery.Te administration of minocycline and deferoxamine in aged mice postsurgery and sleep deprivation showed a notable efect on infammation.Both treatments reduced the levels of proinfammatory cytokines TNF-α and IL-6 compared to the group that did not receive any drug 4 Journal of Clinical Pharmacy and Terapeutics treatment.Te combined drug treatment group displayed the lowest levels of these cytokines.In addition, there was an increase in the anti-infammatory cytokine IL-10 with treatment, suggesting a shift towards a less infammatory state.Te combined treatment group had the highest levels of IL-10, indicating a possible synergistic efect of the two drugs in modulating the infammatory response postsleep deprivation and surgical trauma (Figure 3).

Modulation of Oxidative Stress Markers by Treatment in Aged Mice Postsurgery and Sleep Deprivation.
Te study evaluated the efects of treatments on oxidative stress markers superoxide dismutase (SOD) and malondialdehyde (MDA) in aged mice subjected to sleep deprivation and tibial fracture surgery.Te SOD levels were signifcantly higher in the treatment groups (SM, SD, and SMD) than the TS group at all time points (T1, T2, and T3), indicating an enhanced antioxidant activity.Specifcally, the SMD group showed the highest increase in SOD activity, suggesting a synergistic efect of minocycline and deferoxamine.Conversely, MDA levels, an indicator of lipid peroxidation and thus oxidative stress, were markedly lower in the treatment groups than the TS group.Tis decrease was most pronounced in the SMD group, indicating that the combination of treatments most efectively mitigated oxidative damage.Overall, these results suggest that minocycline and deferoxamine, both individually and in combination, signifcantly modulate oxidative stress markers, improving antioxidant defense and reducing lipid peroxidation in aged mice postsleep deprivation and surgery.Tis indicates a potential protective efect against oxidative stress-related damage in postoperative and sleep-deprived conditions (Figure 4).

Impact of Treatment on CD68 Expression in the Hippocampus of Aged Mice Postsleep Deprivation and Surgery.
Our study assessed the impact of treatments on the expression of CD68, a marker of infammation, in the hippocampus of aged mice subjected to sleep deprivation and tibial fracture surgery.CD68 expression levels were measured at three time points postoperation.Te TS group, which received no treatment postsurgery and sleep deprivation, showed relatively high levels of CD68, with values decreasing slightly over time from 1.48 ± 0.19 at T1 to 1.36 ± 0.17 at T3.In contrast, treatment groups exhibited a signifcant reduction in CD68 expression.Te SM group (minocycline treatment) and SD group (deferoxamine treatment) showed marked decreases in CD68 levels at all time points, indicating a reduction in infammatory activity.Te most signifcant decrease in CD68 expression was observed in the SMD group, which received both minocycline and deferoxamine.Tis group showed the lowest levels of CD68 at all time points, with the most considerable reduction by T3 (0.68 ± 0.14), suggesting a synergistic efect of the combined treatment in reducing infammation.Tese results demonstrate that minocycline and deferoxamine, particularly in combination, efectively reduce infammatory marker CD68 in the hippocampus, which might contribute to mitigating infammation-related damage or cognitive decline associated with surgical trauma and sleep deprivation in aged mice (Figure 5).

Reduction of Hippocampal Iron Content in Aged Mice following Treatment with Minocycline and Deferoxamine.
Te results revealed a signifcant reduction in hippocampal iron content across all treatment groups compared to the sleep deprivation and surgery group (TS).In the TS group, iron content decreased slightly over time from 2.28 ± 0.10 μg/g at T1 to 2.16 ± 0.12 μg/g at T3. Te minocycline treatment group (SM) and the deferoxamine group, and those treated with minocycline (SM group), deferoxamine (SD group), and combined minocycline and deferoxamine (SMD group).Te treatment groups show an increase in crossings compared to the TS group, with signifcant diferences indicated by "A" (P < 0.05 compared to the TS group) and "AB" (P < 0.05 compared to SM and SD groups).(b) Percentage of freezing time in fear conditioning at three postoperative days (PODs) for the same groups.Te SM, SD, and SMD groups exhibit higher freezing percentages than the TS group, indicating improved memory retention.Letters denote statistical signifcance as follows: "A" (P < 0.05 compared to the TS group) and "AB" (P < 0.05 compared to SM and SD groups).(c) Escape latency in the Morris water maze over three postoperative days.All treatment groups showed a decrease in escape latency compared to the TS group, with "A" denoting P < 0.05 compared to the TS group and "AB" denoting P < 0.05 compared to the SM and SD groups. 6 Journal of Clinical Pharmacy and Terapeutics treatment group (SD) showed marked decreases in iron levels at all time points, with the SM group reaching 1.49 ± 0.09 μg/g and the SD group reaching 1.45 ± 0.12 μg/g by T3.Te most signifcant reduction in iron content was observed in the group receiving combined minocycline and deferoxamine treatment (SMD).Tis group showed the lowest levels of iron at all time points, with a substantial decrease to 1.15 ± 0.10 μg/g by T3, indicating that the combination of treatments was most efective in reducing hippocampal iron content (Figure 6).

Efects of Pharmacological Interventions on Iron Regulation and Oxidative Stress in the Hippocampus Postsurgery and Sleep
Deprivation.Related to iron metabolism and oxidative stress in the hippocampal tissue of mice postsurgery and sleep deprivation, the proteins examined were HCP1, TfR, Fpn1, DMT1, hepcidin, and GPX4.Te untreated surgery and sleep deprivation group (TS) displayed baseline expression levels of these proteins.Posttreatment, both minocycline (SM) and deferoxamine (SD) groups showed signifcantly lower expression of HCP1, TfR, Fpn1, DMT1, and Hepcidin, indicating a decrease in iron accumulation and regulation markers.Te combined treatment group (SMD) had the lowest expression of these proteins, suggesting an enhanced efect when both pharmacological agents are used together.For the oxidative stress marker GPX4, the SM and SD groups exhibited an increase in expression, with the SMD group demonstrating the highest expression levels.Tis suggests that the treatments efectively bolster the antioxidative defense mechanisms in the hippocampus (Figure 7).Tese results, reinforced by the Western blot analysis, illustrated that minocycline and deferoxamine, particularly in combination, can modulate    and T3).Tere is a trend of decreasing iron content in all treatment groups compared to the TS group.Te greatest reduction in iron content is seen in the SMD group at all time points, with statistical signifcance indicated by "A" (P < 0.05 compared to the TS group), "AB" (P < 0.05 compared to SM group), and "ABC" (P < 0.05 compared to the SD group).Figure 7: Western blot analysis of iron regulation and oxidative stress proteins in the hippocampus of treated mice.(a) Representative Western blot bands for iron regulation proteins HCP1, transferrin receptor (TfR), ferroportin 1 (Fpn1), divalent metal transporter 1 (DMT1), hepcidin, and oxidative stress protein glutathione peroxidase 4 (GPX4), along with β-actin as a loading control in the TS, SM, SD, and SMD groups.(b-g) Densitometric analysis of HCP1, TfR, Fpn1, DMT1, hepcidin, and GPX4 and normalized to β-actin levels.Te graphs show changes in protein expression levels posttreatment with minocycline (SM), deferoxamine (SD), and their combination (SMD) compared to the untreated surgery and sleep deprivation group (TS).Signifcant decreases in iron regulation proteins are noted in the treatment groups (SM, SD, and SMD) with the lowest expression in the SMD group, while GPX4 expression is increased, indicating enhanced antioxidative response.Signifcance is denoted by "A" (P < 0.05 compared to the TS group), "AB" (P < 0.05 compared to the SM group), and "ABC" (P < 0.05 compared to the SD group).

TS SM SD SMD
the proteins involved in iron homeostasis and oxidative stress, potentially contributing to neuroprotection in the context of surgical trauma and sleep deprivation.

Discussion
Tis study set out to investigate the impacts of sleep fragmentation (SF) on cognitive function in adult and aged mice, with a focus on the role of microglial activation and alterations in iron metabolism and ferroptosis.Te results illuminate the intricate network of processes linking sleep fragmentation, microglial activation, and cognitive impairment, providing novel insights into the pathogenesis of perioperative neurocognitive disorders (PND).Sleep fragmentation was found to signifcantly impact spatial memory and conditioned fear responses in mice, with aged mice displaying increased susceptibility to these efects compared to adult mice.Tis fnding aligns with a growing body of evidence linking sleep disruption to cognitive decline, particularly in older individuals.Sleep is known to play a crucial role in memory consolidation, and disturbances in sleep architecture could interfere with these processes, leading to cognitive impairment.Te fnding that aged mice are more afected by sleep fragmentation than adult mice could be attributed to age-related changes in sleep architecture, increased vulnerability to sleep disturbances, and reduced cognitive reserve in older individuals.
A key fnding in this study was the abnormal activation of microglia due to preoperative sleep fragmentation.Microglia, the resident immune cells of the central nervous system, play pivotal roles in maintaining brain homeostasis and responding to injury or disease.When activated, microglia can release proinfammatory cytokines that can induce neuroinfammation, which has been implicated in cognitive dysfunction.Te alterations in infammatory cytokines observed in this study further support the role of neuroinfammation in sleep fragmentation-induced cognitive impairment.However, the precise mechanisms underlying microglial activation due to sleep fragmentation warrant further investigation.
Another important observation was the alterations in iron metabolism and ferroptosis following sleep fragmentation.Iron is vital for various cellular functions, but excessive iron can lead to the production of reactive oxygen species and oxidative stress, culminating in ferroptosis.Te signifcant reduction in iron content in the hippocampal tissue of mice exposed to sleep fragmentation, coupled with changes in the expression of ferroptosis-related proteins, indicates a link between sleep fragmentation, dysregulated iron metabolism, and ferroptosis.Tese fndings underscore the potential of targeting iron metabolism and ferroptosis as a novel strategy for mitigating sleep fragmentation-induced cognitive impairment.
One limitation of this study is that it was conducted in mice, and the fndings may not be directly translatable to humans due to diferences in sleep architecture and brain structure.Future studies in humans are needed to validate these fndings.Moreover, while this study provides evidence of a link between sleep fragmentation, microglial activation, and cognitive impairment, it does not elucidate the causal relationships between these factors.Further research is needed to dissect these relationships and uncover the underlying mechanisms.
In conclusion, this study sheds light on the complex interplay between sleep fragmentation, microglial activation, and cognitive impairment.It highlights the potential of targeting microglial activation and ferroptosis as preventative strategies for PND in older adults exposed to sleep fragmentation.Tese fndings add to our understanding of the pathogenesis of PND and pave the way for future research aimed at developing efective interventions to improve sleep quality and cognitive outcomes in the perioperative period.

Figure 1 :
Figure 1: Efects of sleep fragmentation (SF) and tibial fracture surgery (TFS) on cognitive function in adult and aged C57BL/6J mice.(a) Number of platform crossings in the Morris water maze for adult mice across four groups: control (AC group), tibial fracture (AT group), sleep deprivation (AS group), and tibial fracture with preoperative sleep deprivation (ATS group).No signifcant diferences are observed.(b) Percentage of freezing time in fear conditioning for adult mice at three time points (T1, T2, and T3) showing minimal diferences between groups.(c) Escape latency in the Morris water maze for adult mice, indicating consistent performance across all groups with a slight increase in the ATS group.(d) Number of platform crossings in the Morris water maze for aged mice, with signifcant decreases in T group, S group, and TS group compared to the C group, and the lowest crossings in the TS group (P < 0.05).(e) Percentage of freezing time in fear conditioning for aged mice, showing a marked reduction in T group, S group, and TS group compared to the C group, especially in the TS group (P < 0.05).(f ) Escape latency in the Morris water maze for aged mice, demonstrating signifcantly increased times in Tgroup, S group, and TS group, with the TS group exhibiting the longest delays (P < 0.05).

Figure 2 :
Figure 2: Efects of minocycline and deferoxamine on cognitive function in aged mice after sleep fragmentation and tibial fracture surgery.(a) Number of platform crossings in the Morris water maze for aged mice in the tibial fracture with preoperative sleep deprivation (TS)group, and those treated with minocycline (SM group), deferoxamine (SD group), and combined minocycline and deferoxamine (SMD group).Te treatment groups show an increase in crossings compared to the TS group, with signifcant diferences indicated by "A" (P < 0.05 compared to the TS group) and "AB" (P < 0.05 compared to SM and SD groups).(b) Percentage of freezing time in fear conditioning at three postoperative days (PODs) for the same groups.Te SM, SD, and SMD groups exhibit higher freezing percentages than the TS group, indicating improved memory retention.Letters denote statistical signifcance as follows: "A" (P < 0.05 compared to the TS group) and "AB" (P < 0.05 compared to SM and SD groups).(c) Escape latency in the Morris water maze over three postoperative days.All treatment groups showed a decrease in escape latency compared to the TS group, with "A" denoting P < 0.05 compared to the TS group and "AB" denoting P < 0.05 compared to the SM and SD groups.

Figure 3 :
Figure3: Infuence of minocycline and deferoxamine on infammatory markers in aged mice postsleep deprivation and surgery.(a) Levels of IL-6 across four treatment groups at three time points (T1, T2, and T3).Te TS group shows the highest levels, while the SM, SD, and SMD groups exhibit reduced IL-6 levels posttreatment.Statistically signifcant diferences are denoted by "A" for P < 0.05 compared to the TS group, "AB" for P < 0.05 compared to the SM group, and "ABC" for P < 0.05 compared to the SD group.(b) Concentrations of IL-10 in the same groups, indicating increased levels in the SM, SD, and SMD groups compared to the TS group, with the SMD group showing the highest levels.Te signifcance indicators are the same as in (a).(c) TNF-α levels displaying a similar trend to IL-6, with all treatment groups showing a decrease compared to the TS group, and the SMD group having the lowest levels.Statistical annotations follow the same pattern as in (a).

Figure 4 :
Figure 4: Efects of treatment on oxidative stress markers in aged mice postsleep deprivation and surgery.(a) Malondialdehyde (MDA) levels, indicating lipid peroxidation, across the TS, SM, SD, and SMD groups at three time points (T1, T2,and T3).MDA levels are lower in the treatment groups than in TS, with the SMD group showing the most signifcant reduction.Signifcance is noted by "A" for P < 0.05 compared to the TS group, "AB" for P < 0.05 compared to the SM group, and "ABC" for P < 0.05 compared to the SD group.(b) Superoxide dismutase (SOD) activity levels in the same groups.Elevated SOD activity is observed in the treatment groups, with the highest levels in the SMD group, indicating increased antioxidant capacity.Statistical annotations are consistent with those in part (a).

Figure 5 :Figure 6 :
Figure 5: Changes in CD68 expression in the hippocampus of aged mice following treatment postsleep deprivation and surgery.(a) Representative Western blot bands for protein CD68 along with β-actin as a loading control in the TS, SM, SD, and SMD groups.(b)Expression of CD68, normalized to β-actin, in the hippocampus of aged mice across four groups: tibial fracture with preoperative sleep deprivation (TS group), minocycline treatment (SM group), deferoxamine treatment (SD group), and combined minocycline and deferoxamine treatment (SMD group) at three postoperative time points (T1, T2, and T3).Te TS group shows the highest expression levels, with a slight downward trend over time.Te SM, SD, and SMD groups all exhibit signifcant reductions in CD68 expression compared to the TS group, with the SMD group showing the greatest decrease, especially by T3.Statistical signifcance is denoted by "A" (P < 0.05 compared to the TS group), "AB" (P < 0.05 compared to the SM group), and "ABC" (P < 0.05 compared to the SD group).