Cigarette smoke-induced pulmonary impairment is associated with social recognition memory impairments and alterations in microglial profiles within the suprachiasmatic nucleus of the hypothalamus

Chronic obstructive pulmonary disease (COPD) is a major, incurable respiratory condition that is primarily caused by cigarette smoking (CS). Neurocognitive disorders including cognitive dysfunction, anxiety and depression are highly prevalent in people with COPD. It is understood that increased lung inflammation and oxidative stress from CS exposure may ‘spill over ’ into the systemic circulation to promote the onset of these extra-pulmonary comorbidities, and thus impacts the quality of life of people with COPD. The precise role of the ‘spill-over ’ of inflammation and oxidative stress in the onset of COPD-related neurocognitive disorders are un-clear. The present study investigated the impact of chronic CS exposure on anxiety-like behaviors and social recognition memory, with a particular focus on the role of the ‘spill-over ’ of inflammation and oxidative stress from the lungs. Adult male BALB/c mice were exposed to either room air (sham) or CS (9 cigarettes per day, 5 days a week) for 24 weeks and were either daily co-administered with the NOX2 inhibitor, apocynin (5 mg/kg, in 0.01 % DMSO diluted in saline, i.p.) or vehicle (0.01 % DMSO in saline) one hour before the initial CS exposure of the day. After 23 weeks, mice underwent behavioral testing and physiological diurnal rhythms were assessed by monitoring diurnal regulation profiles. Lungs were collected and assessed for hallmark features of COPD. Consistent with its anti-inflammatory and oxidative stress properties, apocynin treatment partially lessened lung inflammation and lung function decline in CS mice. CS-exposed mice displayed marked anxiety-like behavior and impairments in social recognition memory compared to sham mice, which was prevented by apocynin treatment. Apocynin was unable to restore the decreased Bmal1-positive cells, key in cells in diurnal regulation, in the suprachiasmatic nucleus of the hypothalamus to that of sham levels. CS-exposed mice treated with apocynin was associated with a restoration of microglial area per cell and basal serum corticosterone. This data suggests that we were able to model the CS-induced social recognition memory impairments seen in humans with COPD. The preventative effects of apocynin on memory impairments may be via a microglial dependent mechanism.


Introduction
Chronic obstructive pulmonary disease (COPD) is currently the 3 rd leading cause of death globally, affecting 210 million people worldwide with cigarette smoke (CS) exposure being the major cause in industrialized countries (Vogelmeier et al., 2017). Noxious particles in CS causes lung epithelial damage and induces an inflammatory response involving the accumulation of macrophages, neutrophils and lymphocytes as well as the release of pro-inflammatory mediators inducing further recruitment of leukocytes and a vicious cycle of persisting lung inflammation (Barnes et al., 2003;Vlahos and Bozinovski, 2014;Bozinovski et al., 2015). Overexuberant accumulation of pulmonary macrophages and neutrophils results in elevated reactive oxygen/nitrogen species (ROS/ RNS) production, increasing the oxidative burden in addition to the ROS/RNS already present in CS (Rahman et al., 1996;Drost et al., 2005;Tuder et al., 2003;Bernardo et al., 2015). The increased pulmonary inflammation and oxidative stress in response to CS observed in COPD lungs may 'spill over' into the systemic circulation driving the manifestation of additional chronic medical conditions (also known as comorbidities), such comorbidities include cognitive decline and mood disorders such as anxiety and depression (Barnes and Celli, 2009).
Cognitive decline is present in up to 61% of people with COPD whilst anxiety and depression have an estimated prevalence between 40-64 % and 25-67 %, respectively (Vikjord et al., 2020;Heslop-Marshall et al., 2018;Dodd, 2015). Moreover, these neurocognitive comorbidities can adversely affect patient outcomes such as increased morbidity, disability, healthcare expenditure (including but not limited to increased length of hospital stay, readmission rates, and increase rate of acute exacerbations) and mortality (Maurer et al., 2008;Yohannes et al., 2018;Li et al., 2019). Vikjord and colleagues demonstrated that people with COPD showing symptoms of anxiety had higher cumulative smoking exposure (Vikjord et al., 2020). However, others have found no clear associations between smoking and anxiety or self-medicatinginduced smoking in anxious populations. Despite the high prevalence, neurocognitive comorbidities are often under diagnosed and under treated resulting in adverse outcomes in patients (Tselebis et al., 2016). This is largely due to the lack of understanding on the underlying mechanisms driving COPD-related neurocognitive dysfunction and the lack of accurate diagnostic tools. In the non-COPD setting, anxiety-and depressive-like behaviors have been linked to alternations in the core transcriptionaltranslational feedback loops that are involved in diurnal rhythm regulation.
Endogenous circadian rhythm allows for synchronisation of physiological and behavioural processes over ~24 hours by core circadian clock genes within the suprachiasmatic nucleus (SCN) of the hypothalamus (Takahashi, 2015;Harbour et al., 2014). The primary transcriptionaltranslational feedback loop consists of a Circadian Locomotor Output Cycle Kaput (CLOCK) and Brain and Muscle ARNT-like protein-1 (BMAL1) heterodimer complex in the cytosol of SCN cells. The CLOCK: BMAL1 complex translocates to the nucleus activating the expression of Period (PER1/2/3) and Cryptochrome (CRY1/2) proteins forming a heterodimer complex and repressing their own transcription by acting on the CLOCK:BMAL1 complex (Takahashi, 2015;Harbour et al., 2014). In nocturnal rodents (i.e., mice and rats), CLOCK:BMAL1 activation commences during the active period (i.e., dark phase) whilst PER:CRY protein transcription occurs during the less-active period (i.e., light phase) inducing the repression of CLOCK:BMAL1 transcription in the evening (Takahashi, 2015;Harbour et al., 2014). Circadian clock disruption has been strongly implicated in the pathophysiology of mood disorders and cognitive decline. Clinical studies demonstrate that major depressive disorder is correlated with misalignment of diurnal rhythms (Emens et al., 2009) alongside altered expression profiles of core circadian clock genes in post-mortem brains of people with major depressive disorder (Li et al., 2013). Similarly, studies with rodents have demonstrated an association between altered diurnal profiles and anxiety-like behaviors. Genetic disruption of circadian rhythm via Per1 and Per2 gene knockdown induced elevated anxiety-like behavior and impaired spatial working memory (Spencer et al., 2013;Kim et al., 2022), whilst mutations in the Clock gene has been shown to be linked to extreme manic-like behaviors (Arey et al., 2014). Interesting, deletion of Per2 in glial cells, but not neuronal cells, in mice leads to reduced anxiety-like behavior. However, the authors suggest that this is not a defective molecular clock, as glial Bmal1 deletion had no effect (Martini et al., 2021).
Chronic CS exposure is a recognised form of environmental perturbations that has been shown to alter diurnal rhythmicity in peripheral circadian clocks. Literature suggests that people with COPD experience worsened symptoms and severity of the disease in the early morning or late night including reduced lung function (Tsai et al., 2007;Scichilone et al., 2019), nocturnal oxygen desaturation (McNicholas et al., 2013), and cardiac arrhythmias (McNicholas et al., 2013). In rodents, acute and chronic exposure to CS induced alterations in expression of core clock genes resulting in enhanced lung inflammation and altered lung function (Hwang et al., 2014;Wang et al., 2021). Despite the accumulating evidence that lung clock rhythmic expression is altered in both people with COPD and preclinical models of COPD, the contribution to extra-pulmonary comorbidities such as cognitive decline and anxiety remains under investigated. Therefore, we evaluated the effect of CS exposure on both cognitive and anxiety-like behaviors and core clock expression profiles in the SCN of the hypothalamus. It is well established that increased oxidative burden dysregulates diurnal rhythm (reviewed in (Wilking et al., 2013)). We have previously shown that apocynin, a NADPH oxidase-2 (NOX2) inhibitor that acts as a scavenger for superoxide (O 2 -) and other ROS molecules (Heumu¨ller et al., 2008), and can simultaneously attenuate lung inflammation and oxidative stress in murine models of COPD and acute exacerbations of COPD (Duong et al., 2010;Oostwoud et al., 2016). Thus, we also examined if co-administration of apocynin and CS exposure is sufficient to attenuate lung inflammation and prevent neurocognitive behaviors following CS exposure.

Animals
We conducted all experiments in accordance with the Australian National Health and Medical Research Council's (NHMRC) Code of Practice for the Care and Use of Animals for Scientific Purposes, with approval from the RMIT University Animal Ethics Committee (AEC1928). For these experiments, specific pathogen-free male BALB/c mice obtained from the Animal Resource Centre (Perth, WA, AUS) arrived at 7 weeks of age. The mice were kept under standard laboratory housing conditions, with a 12 h light cycle (7am to 7pm), an ambient temperature of 22 • C, with humidity between 40-60 %, in 250 lx lighting, and free access to water and standard mice chow.

Cigarette smoke exposure
After a one-week of acclimatization period, mice were randomly assigned to four different groups (n = 16 per group) that were matched for body weight. Mice were exposed to CS or room air (sham) for 24weeks with co-administration of either apocynin (5 mg/kg, 0.01% DMSO in sterile saline, intraperitoneal [i.p.]) or vehicle (0.01% DMSO in sterile saline, i.p.) as previously described (Vlahos et al., 2006;Chan et al., 2020;Hepworth et al., 2019). Treatments were administered via i. p. injection once daily, one hour prior to the initial CS exposure. Briefly, mice were placed inside an 18-liter plastic chamber and underwent whole body exposure to the smoke of 1 filtered cigarette (Winfield Red, 16 mg or less of tar, 1.2 mg or less of nicotine and 15 mg or less of CO, Phillip Morris, VIC, AUS) over 15 min with a 5 min recovery interval where mice were exposed to room air. This was then repeated such that the mice received 3 cigarettes over a one-hour period and repeated three times a day (8 am, 12 pm, and 4 pm), thus, the mice were exposed to 9 cigarettes/day for 5 days (Monday-Friday) a week. This exposure protocol generates carboxyhemoglobin levels within the range observed in human smokers. Smoke was generated in 60 mL tidal volumes over 10 s by use of timed draw-back mimicking normal smoking inhalation volume and cigarette burn rate. The mean total suspended particulate mass concentration in the chamber containing CS generated from one cigarette was 419 mg/m 3 . Sham animals were handled identically without CS exposure. Following 23 weeks of CS or air exposure, a cohort of mice underwent neurocognitive behavioral testing, and diurnal rhythms parameters were captured using an indirect calorimetry system (Comprehensive Laboratory Animal Monitoring System; CLAMS, Columbus Instruments, OH, USA), as described below and in Fig. 1.

Anxiety-like behavioral tests
To assess the impact of CS exposure on neurocognitive behaviors, we tested the mice in an open field task with or without novel objects (n = 12 per group), and the social interaction task (n = 5 per group). To prevent the onset of withdrawal phenotype, neurocognitive testing was performed between Tuesday and Friday following 23 weeks of CS exposure. All anxiety-like behavioral experiments were completed between 9 am and 1 pm to limit potential effects of diurnal rhythms on any parameters measured.
Open Field: Mice were placed in the center of an empty open field arena (a black plywood box, 65 cm × 65 cm × 65 cm) for 8 mins. Mice were filmed, and an experimenter blinded to treatment groups scored the videos using Ethovision 15 (Noldus Information Technology, Wageningen, The Netherlands). Using Ethovision, zones were created that corresponded to the center of the arena and the remaining space (edge). Mice were scored for the total distance moved and duration spent in the center (s).
Object Exploration in the Open Field: Following habituation in the open field task, we examined how mice would respond to novelty exposure (assesses neophobia which is used as an indicator of anxiety). Briefly, we used the same experimental test settings and conditions used in the open field. Mice were placed in the center of the arena and allowed to explore two identical objects placed equally distanced apart for 8 mins. Mice were filmed, and an experimenter blinded to treatment groups scored the videos using Ethovision 15 (Noldus Information Technology, Wageningen, The Netherlands). Using Ethovision, zones were created that corresponded to the two identical objects and were scored for the total distance moved (m) and duration spent exploring the objects (s).
Three-Chambered Social Interaction Test: The three-chambered social interaction test was performed to assess sociability and social novelty, as described previously (He et al., 2018;Yan et al., 2020). The sociability apparatus consisted of a transparent Perspex box (60 cm × 40 cm × 22 cm) divided into three chambers using transparent Perspex walls with a slide door (5 × 8 cm) at the bottom, opening from the central compartment. The wire cages (15 cm [height] × 7 cm [diameter] with grey PVC top and bottom lids with stainless steel grid bars) allows for mice to interact closely with the novel caged mouse. Mice were placed in the center chamber and allowed to habituate to the chamber for 5 mins.
The chamber doors were opened and mice were allowed to explore the two empty wire cages for 7 mins. Mice were then carefully placed back into the center chamber and a novel mouse (stranger 1) was placed in one of the wire cages, while the other remained empty. The chamber doors were opened and the subject mouse was allowed to freely explore all three chambers for 8 mins. The subject mouse was then returned to the center chamber, and a novel mouse (stranger 2) was placed into the previously empty wire cage. Again, the chamber doors were opened and the subject mouse was allowed to freely explore all three chambers for 8 mins. The positions of the stranger mouse was alternated between tests. Each mouse was filmed, and an experimenter double blinded to treatment groups manually scored the time spent interacting/exploring each stimulus. The sociability index was calculated as the ratio of time spent exploring stranger 1 minus the time spent exploring the empty wire cage divided by the total exploration time. The social preference index was calculated as the total amount of time spent exploring stranger 1 or stranger 2 divided by the total exploration time.

Indirect calorimetry
To assess the impact of CS exposure on diurnal rhythm and energy metabolism, a cohort of the mice were placed into the CLAMS, immediately after the final CS exposure of the day. After 4 h of acclimatization, data collection began, providing activity recordings at 20 min intervals, and continuing for a further 48 h. Standard chow was provided ad libitum throughout the experiment. The system monitors total activity, oxygen consumption (VO 2 ), respiratory exchange ratios (RER; substrate utilization) and exergy expenditure. For substrate utilization, an RER value closer to 1.0 represents carbohydrate as the predominant energy source which is typical during feeding. Meanwhile, an RER value closer to 0.6 denotes lipid oxidation as the predominant energy source. Mean data are presented over a 24-hour period as well as 12-hour blocks representing the light and dark cycle. Timeline of experimental design. Mice were exposed to room air (sham) or cigarette smoke (CS; 9 cigarettes/day, 5 days/week) with co-administration of either apocynin (5 mg/kg, 0.01 % DMSO in sterile saline, i.p.) or vehicle (0.01 % DMSO in sterile saline, i.p.) for 24 weeks. Neurocognitive and diurnal energy regulation assessments were performed on mice one week prior to protocol conclusion (week 23). Cohort 1: To examine the inflammatory profile within the lungs, we collected bronchoalveolar lavage (BAL) fluid for BAL differentiation and dissected the lungs for qPCR. The whole brain was dissected for immunofluorescence. Cardiac blood was collected for corticosterone analysis. Cohort 2: Immediately prior to the cull, lung function assessment was performed (week 24). To examine the circadian rhythm profile within the hypothalamus, we dissected the left and right hypothalamic for qPCR.

Lung cellular inflammatory response
One week after the anxiety-like behavior and indirect calorimetry assessment, mice were sacrificed; between 9 am and 10 am, with an i.p injection of sodium pentobarbital (150 mg/kg). To assess lung inflammation, lungs of terminally anaesthetized mice (cohort 1; Fig. 1) were lavaged in situ with 0.4 mL phosphate-buffered saline (PBS; 4 • C, pH 7.4), followed by three 0.3 mL of PBS, with approximately 1 mL of bronchoalveolar lavage fluid (BALF) recovered from each animal (n = 16 per group). BALF cytospins were prepared at 400 rpm for 10 min on a Shandon Cytospin 3 Cytocentrifuge (Akribis Scientific Limited, Cheshire, UK). Cytospin slides were fixed with Kwik-Diff fixative (Thermo Fisher Scientific), then stained with Hemacolor Rapid Stain Solutions 2 and 3 (Merck Millipore, Burlington, MA, USA), and 500 cells per slide were counted and differentiated into macrophages, neutrophils and lymphocytes using standard morphological criteria. The lungs were then perfused with 10 mL of PBS to remove blood, rapidly excised en bloc, blotted and the large left lobe was snap frozen in liquid nitrogen and stored at − 80 • C for quantitative real-time-PCR (qRT-PCR) analysis.

Lung function testing
To assess pulmonary function following chronic CS exposure and apocynin treatment, a separate cohort of mice from each group (n = 8 per group; cohort 2 in Fig. 1) underwent lung function testing using a small-animal FlexiVent ventilator system (Scireq, Montreal, QC, Canada) between 8:30 and 10.30 am (15.5-17.5 h after the last CS exposure) as previously described . Following anaesthetisation (15 mg/kg ketamine and 1.5 mg/kg xylazine i.p.), a tracheostomy was performed using an 18G cannula, with a suture sealing the wall of the trachea around the cannula. The mouse was then connected to the FlexiVent system, assessing different respiratory parameters including force oscillation, deep inflation, and constant phase models in mice with a breathing rate of 150 breaths per min. Deflation of the lungs via Negative Pressure Forced Expiration was performed to extract forced vital capacity levels. To assess changes in respiratory capacity, we generated pressure-volume (PV) loops by slowing inflating the lungs via Positive-End Expiratory Pressure and then deflated to generate a partial PV loop and derive the static compliance (Cst) and inspiratory capacity (IC) levels.

Serum corticosterone
To assess basal corticosterone concentrations from mice in cohort 1, blood was collected via the inferior vena cava (16 h after the last CS exposure) into a Microvette 500 Serum Gel tube (Sarstedt, Germany) and left to clot for at least 15 min. Whole blood samples were centrifuged at 10,000 g for 5 min at room temperature (RT). Serum was then aspirated into fresh tubes and stored at − 80 • C until required. We used a standard mouse corticosterone enzyme-linked immunosorbent assay (ThermoFisher Scientific, Waltham, MA, USA). The inter-assay variability for the assay was 7.9 % coefficient of variation (CV), intra-assay was 5.2 % and the lower limit of detection was 78.125 pg/mL. Samples from all treatment groups were assayed together in duplicates.

Brain dissections
The brains from mice that underwent lung function testing were sagitally hemisected through the midline (cohort 2; Fig. 1). The hypothalamus was dissected over ice and samples were immediately snapfrozen in liquid nitrogen and stored at − 80 • C (n = 8 per group). The brains of mice from cohort 1 (n = 8 per group) were immersion fixed in 4 % paraformaldehyde in PBS (4 • C, pH 7.4) for 24 hours before cryoprotecting in 20 % sucrose for at least 24 hours. All brain tissue was collected between 9 am and 10am to allow consistent assessment of circadian rhythm assessment. The brains were then cut into 30 µM coronal sections in a one-in-five series using a cryostat and stored at 4 • C until use.

Gene expression
The lungs and hypothalamus collected above were used to determine changes in candidate genes involved in pro-inflammatory profiles and diurnal rhythms, respectively. RNA was isolated using a RNeasy Mini and a RNeasy Lipid Tissue Mini purification kit respectively (QIAGEN, Valencia, CA, USA). RNA (1 µg) was transcribed to complementary cDNA using the High-Capacity RNA-to-cDNA Kit (ThermoFisher Scientific), following the manufacturer's instructions. We performed qRT-PCR using Taqman Gene Expression Assays (ThermoFisher Scientific) on QuantStudio 7 Flex instrument. All reactions were performed in triplicate, and we compared a relative quantitative measure of the target gene expression with an endogenous control, Rps18 (see Table 1). We analyzed mRNA expression using the equation 2 -ΔΔC(t) , where C(t) is the threshold cycle at which fluorescence is first detected significantly above background. Data are presented as a fold increase relative to sham vehicle.

Immunohistochemistry
To determine the effect of CS exposure on diurnal rhythm proteins, we immunolabelled brain sections for Bmal1 in the SCN of the hypothalamus, a core region involved in diurnal rhythm regulation. The antibody has been verified by the manufacturers, by other researchers, and in our hands with the use of primary and secondary negative controls. Sections containing SCN of the hypothalamus were selected from each animal and representatives from each treatment group were processed at the same time. We blocked the sections for 1 hour at RT with 3 % BSA, 4 % NHS, 0.3 % Triton X-100 in PBS-T. The sections were then incubated in primary antibody (1:800, rabbit, Novus Biologicals, Littleton, CO, USA) for 48 hours at 4 • C. Following the incubation period, sections were washed 3 × 10 min in PBS-T to remove excess primary antibody and then incubated in secondary antibody (1:200, biotinylated anti-rabbit, Vector Laboratories, Burlingame, CA, USA) for 2 hours. Sections were washed in PBS-T followed by avidin-biotin horseradish peroxidase (HRP) complex (ABC; 1:200, Vector Laboratories) for 45 mins. Sections were then incubated in diaminobenzidine with nickel and cobalt to visualise the HRP activity. The reaction was stopped when there was optimal contrast between specific cellular and non-specific background labelling. Sections were then mounted onto slides, air dried, dehydrated in a series of alcohols, cleared in histolene, and coverslipped in Entellan™ new (Merck Millipore). Photomicrograph images of the SCN of the hypothalamus were taken on an upright Olympus BX53 microscope (Olympus Corp., Tokyo, Japan) using a 20x objective and CELLSENS life science imaging software (Olympus Corp.). All photomicrographs were assessed by a researcher blinded to treatment groups and cells were manually counted. We analysed three sections 60 µm apart between 0.34 and 0.82 mm relative to the bregma. The sum of the counts was used as our sampled result.

Immunofluorescence
To determine the effect of CS exposure on glial cells (astrocytes and microglia), sections containing the SCN of the hypothalamus were immunolabelled for Glial Fibrillary Acidic Protein (GFAP) and Ionized Calcium-Binding Adapter Molecule-1 (Iba-1). Sections through the SCN of the hypothalamus were selected from each animal and representatives from each treatment group were processed at the same time. Sections were blocked for 2 hours at RT with 3 % BSA, 4 % NHS, 0.3 % Triton X-100 in PBS-T before submerging the sections in the primary antibody overnight at 4 • C (GFAP: 1:500, anti-rabbit [#1022-5, Dako, Bath, UK] and Iba-1: 1:1000, anti-rabbit [#Z0334, Fujifilm Wako, Osaka, Japan]). Following the primary incubation, the sections were transferred into the secondary antibody (2 hours, 1:500, Alexa Fluor 488 goat anti-rabbit, Thermo Fisher). Sections were counterstained with Fluoromount-G TM , with DAPI (Thermo Fisher Scientific) and imaged using the 20x objective on the upright Olympus BX53 microscope (Olympus). All photomicrographs were assessed by an experimenter blinded to treatment groups and Iba-1-and GFAP-positive cell density was analysed using imaging software cellSens Dimension by thresholding the positive stains against the background.

Data analysis
All data was analyzed using a two-way analysis of variance (ANOVA). Where significant interactions were found, we then performed Tukey post hoc tests. Data are presented as the mean + SEM. Statistical significance was assumed when p ≤ 0.05.

Apocynin treatment attenuated lung inflammation in CS-exposed mice
Six months of CS exposure caused a reduced body weight gain compared to sham mice (interaction between day + exposure and treatment: F (72,648) = 7.199, p < 0.0001; Fig. 2A, B). Apocynin treatment significantly increased body weight gain in sham mice compared to sham vehicle mice. Mice exposed to CS and apocynin treatment significantly increased weight gain compared to CS vehicle mice but was unable to restore weight to sham levels ( Fig. 2A, B). This weight loss was associated with an 8.03-fold increase in total cell counts in CS vehicle mice compared to sham vehicle mice (Fig. 2C) and this was mainly attributed to a marked increase in the number of macrophages (4.97fold increase; Fig. 2D), neutrophils (123.12-fold increase; Fig. 2E) and lymphocytes (23.55-fold increase; Fig. 2F). Apocynin treatment partially attenuated the increased total cellularity (6.34-fold increase; interaction between exposure and treatment: F (1,15) = 8.814, p = 0.0096; Fig. 2C) which was mainly attributed to a significant reduction in the number of macrophages in BALF compared to sham vehicle (3.53fold increase; interaction between exposure and treatment: F (1,15) = 12.62, p = 0.0029; Fig. 2D) without detectable effects on the neutrophils and lymphocytes counts.
To further characterize the inflammatory profile following apocynin administration, we evaluated the mRNA expression in whole lung tissue. CS-exposure caused a significant increase in the mRNA expression of Tnfa in the lung of CS vehicle-exposed mice when compared to sham mice and apocynin treatment resolved the Tnf expression to sham levels (interaction between exposure and treatment: F (1,24) = 8.23, p = 0.0085; Fig. 2G). Apocynin treatment reduced the expression levels of Il6 and Il1b following CS exposure compared to CS Vehicle mice (Il6: interaction between exposure and treatment: F (1,23) = 8.71, p = 0.0071; Fig. 2H and Il1b: interaction between exposure and treatment: F (1,23) = 7.71, p = 0.010; Fig. 2I). Gene expression of the superoxide generating enzyme, Nox2 (Cybb), was significantly increased following CS vehicle exposure compared to sham vehicle mice (interaction between exposure and treatment: F (1,24) = 9.22, p = 0.0057; Fig. 2J) which was restored to sham vehicle levels in CS apocynin mice. Mmp12 expression was significantly elevated in CS vehicle mice compared to sham vehicle mice and this was partially restored following a treatment with apocynin, however, not to sham mice levels (interaction between exposure and treatment: F (1,24) = 5.98, p = 0.0227; Fig. 2K).

Apocynin treatment partially preserved lung dysfunction in CSexposed mice
Chronic CS exposure caused reduced elastic recoil (static compliance; Fig. 3A), hyperinflation (inspiratory capacity; Fig. 3B), increased air exhalation (forced vital capacity; Fig. 3C) and alveolar collapse (increase PV loop area; Fig. 3D, E) compared to sham vehicle mice. Apocynin was unable to prevent the loss in elastic recoil by CS exposure as evident by a significant increase in static compliance (interaction between exposure and treatment: F (1,7) = 6.494, p = 0.0382; Fig. 3A), but attenuated lung hyperinflation as demonstrated by a reduced inspiratory capacity compared to CS vehicle mice (interaction between exposure and treatment: F (1,7) = 12.85, p = 0.0089; Fig. 3B). Apocynin was able to partially preserve the CS-induced increase in PV loop area (interaction of exposure by treatment: F (1,7) = 7.596, p = 0.0283, Fig. 3D), however, apocynin did not prevent the upward shift of the PV loop curve in CS-exposed mice indicative of alveolar collapse (Fig. 3E). Apocynin was also unable to preserve the amount of air being exhaled (increased forced vital capacity) in CS mice (interaction between exposure and treatment: F (1,7) = 11.93, p = 0.0106; Fig. 3C).

Apocynin improves CS-induced social recognition memory impairments
To investigate whether chronic CS-induced lung inflammation and dysfunction was associated with changes in both cognitive and anxietylike behavior, we assessed performance in three behavioral tasks. As seen in Fig. 4A, B, CS-exposure did not alter locomotor activity in the open field, nor time spent in the center of the arena compared to sham vehicle mice (Fig. 4C). Sham apocynin mice spent more time in the center of the arena compared to sham vehicle mice but this was reduced in apocynin treated CS-exposed mice (interaction of exposure by treatment: F (1,41) = 6.66, p = 0.0013; Fig. 4C, D). We next sought to assess aversion to novelty (i.e. object neophobia) in the open field immediately after the open field test. Similar to the open field, CS-exposure did not alter locomotor activity in this task compared to sham mice (Fig. 4 E, F). However, CS vehicle-exposed mice spent significantly less time investigating the novel objects in the arena compared to sham vehicle mice suggesting that CS exposure induces neophobia-like responses to changes in the environment. A treatment with apocynin was unable to restore exploration time in mice exposed to CS (main effect of exposure: F (1,44) = 6.242, p = 0.0013 and main effect of treatment: F (1,44) = 25.17, p < 0.0001; Fig. 4G, H).
To examine sociability, a stranger mouse (stranger 1) was placed into one of the chambers and an empty wire cage into the other chamber and the test mouse was freely allowed to explore the apparatus. In this phase, sham and CS mice spent significantly more time with the stranger 1 compared to empty wire cage (main effect of novelty: F (1,32) = 44.28, p < 0.0001 and main effect of treatment and exposure: F (3,32) = 5.037, p = 0.0057; Fig. 5A, B), however, we did not show any differences in sociability index between sham and CS exposure (Fig. 5C). To examine the effect of CS exposure and apocynin treatment on social novelty, a second stranger mouse (stranger 2) was placed in the empty wire cage. We found no differences in total exploration time of test mice for both stranger 1 and stranger 2 between groups (Fig. 5D). When assessing social preference index, sham vehicle and sham apocynin showed a preference for stranger 2, however, CS vehicle mice displayed a neutral preference (50 % preference index) for both stranger 1 and stranger 2 suggesting exposure to CS leads to social recognition memory impairments (interaction of exposure + treatment by novelty: F (3,32) = 15.85, p < 0.0001, Fig. 5E). CS exposure plus apocynin treatment for 24-weeks resulted in increased interactions with the novel mouse (stranger 2) comparable to that of sham mice. Overall, our findings suggest that CSexposure did not alter the anxiety-like behaviors in the open field, however, these mice showed a significant aversion to novelty in the open field arena. These mice also displayed social recognition memory impairments in the sociability test and apocynin restored this impairment in CS exposed mice to sham levels.

Apocynin did not restore the disrupted diurnal energy regulation profile by CS exposure
Recent studies suggest that diurnal rhythm disruption may evoke impairments in cognitive and anxiety-like behaviors, thus, we sought to examine whether CS-exposure disrupts diurnal energy regulation and whether inhibition of oxidative stress, via apocynin treatment, could restore diurnal energy regulation profiles using indirect calorimetry. Regardless of treatment, locomotor activity was increased during the light and decreased during the dark phase in CS-exposed mice compared to sham equivalents (interaction between exposure and treatment: F (3,40) = 13.10, p < 0.0001; Fig. 6A, B). Strikingly, a sharp forward spike in total activity was consistently observed (at ~ 18:00) in the CS-exposed mice compared to sham mice (occurring at ~ 19:00), suggesting a shift in diurnal energy regulation. This reduced activity during the dark phase was associated with a significant increase in the VO 2 levels during the light phase in CS exposed mice, irrespective of treatment (main effect of exposure and treatment: F (3,40) = 12.04, p = 0.0001; Fig. 6C, D). In line with this data, the RER was also reduced during the dark phase in CS vehicle mice compared to sham mice, indicating that these mice were relying on lipid oxidation as fuel rather than carbohydrates such as seen in the sham mice (interaction between exposure and light cycle: F (3,40) = 12.59, p < 0.0001; Fig. 6E, F). Moreover, this shift in total activity and metabolic profiles was associated with a reduction in energy expenditure illustrating that the CS-exposed mice were consistently maintaining light phase energy expenditure levels during the dark phase (main effect of light phase: F (1,40) = 25.98, p < 0.0001; Fig. 4G, H). Overall, CS exposure disrupted diurnal energy regulation which persisted despite apocynin administration.

Disruption in diurnal energy regulation is linked to a change in circadian rhythm profile in the SCN of the hypothalamus
We next sought to examine whether the disrupted diurnal energy regulation may be linked to changes in genes and proteins in the SCN of the hypothalamus which regulates diurnal rhythm and anxiety-like behaviors at a single timepoint (9am). In the SCN of the hypothalamus, we found a main effect of treatment in Bmal1 (Arntl) gene expression with a decreased expression following CS vehicle exposure whilst CS apocynin mice displayed an increasing trend (p = 0.050) towards significance  (F (1,28) = 4.36, p = 0.0420; Fig. 7A). Mice exposed to CS and apocynin displayed a significant increase in Clock and Nr1d1 gene expression compared to sham apocynin mice (Clock: interaction between exposure and treatment: F (1,28) = 16.02, p = 0.0004; Fig. 7B and Nr1d1: interaction between exposure and treatment: F (1,28) = 4.750, p = 0.0379; Fig. 7C). Under normal physiological conditions, following the activation of the Clock:Bmal1 heterodimer complex, the complex binds to Dbp to activate the transcription of Per1 and ultimately the Per:Cry heterodimer complex. CS vehicle exposure significantly increased Dbp expression compared to sham vehicle mice and this was not prevented by apocynin (main effect of exposure: F (1,28) = 51.18, p < 0.0001; Fig. 5D). We found no difference between sham and CS vehicle mice for Cry1, Cry2 and Per1. However, CS exposure mice co-treated with apocynin displayed a significant increase in Cry2 and Per1 gene expression compared to sham apocynin mice (Cry2: interaction between exposure and treatment: F (1,28) = 9.632, p = 0.0043; Fig. 7F and Per1: main effect of exposure: F (1,28) = 10.59, p = 0.0030 and main effect of treatment: F (1,28) = 17.81, p = 0.0002; Fig. 7G). CS exposure increased Per2 gene expression compared to sham vehicle mice and apocynin treatment further augmented this expression (interaction between exposure and treatment: F (1,28) = 11.31, p = 0.0022; Fig. 7H).
CS vehicle mice had a significantly reduced number of Bmal1positive cells in the SCN compared to sham vehicle mice (interaction between exposure and treatment: F (1,32) = 5.886, p = 0.0211; Fig. 7I, J). Moreover, apocynin co-administration did not prevent this reduction in Bmal1-positive cells by CS exposure. These changes in core diurnal rhythm-regulating genes and protein, at an individual timepoint, in combination with the altered diurnal energy profile may indicate that there is a disruption in diurnal rhythm profiles by CS exposure, however, additional timepoints are required to definitely discern this.

Fig. 5.
Apocynin reverses chronic cigarette smoke (CS) exposure-induced social recognition memory impairments in mice. Behavioral testing was performed on mice 1 week prior to protocol conclusion. (A) Schematic representation of the social interaction test (SIT), (B) time spent (seconds) exploring the empty wire cage and stranger 1 in the habituation phase of the SIT task, (C) Sociability Index in the habituation phase of the SIT task (n = 5 per group), (D) time spent (seconds) exploring the stranger 1 (familiar) and stranger 2 (novel) in the sociability phase of the SIT task and (E) social preference index in the sociability phase of the SIT task. Data are expressed as mean + SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. E: Red line denotes significantly different from chance.

Apocynin is able to preserve microglial but not astrocyte profiles following CS exposure
Astrocytes and microglia in the SCN of the hypothalamus are rapidly emerging as key cellular contributors modulating neurocognitive behaviors (Vadnie et al., 2022). Thus, we assessed the impacts of CSexposure on glial profiles in the SCN of the hypothalamus. CSexposure led to an increased astrocyte density in the SCN of the hypothalamus compared to their sham counterparts, and this was unaltered by apocynin co-administration (interaction between exposure and treatment: F (1,28) = 6.847, p = 0.0142; Fig. 8A, D). We also saw that chronic CS-exposure increased the area per cell of Iba-1 positive cells compared to sham vehicle mice (interaction between exposure and treatment: F (1,26) = 19.51, p = 0.0002; Fig. 8B, E). Strikingly we also found that apocynin treatment in mice exposed to room air (sham) increased the area per cell compared to sham vehicle mice. In CS exposed mice, apocynin was able to restore the Iba-1 positive cell profile to sham vehicle levels.

Apocynin co-administration restores CS-induced corticosterone levels
Hypersecretion of corticosterone is known to confer vulnerability to mood disorders. Elevation of serum corticosterone levels also marks potential dysregulation of the hypothalamic-pituitaryadrenal (HPA) axis. Thus, we assessed whether there were differences in serum corticosterone levels. CS exposure reduced serum corticosterone levels compared to sham vehicle and sham apocynin mice and this was resolved following apocynin treatment (main effect of exposure: F (1,20) = 10.67, p = 0.0039; Fig. 8C). Fig. 6. Apocynin treatment is unable to restore diurnal regulation profiles following cigarette smoke (CS) exposure. A cohort of mice were placed into the CLAMS to assess diurnal rhythm and energy metabolism. (A) Total activity levels (n = 6 per group), (B) average total activity levels, (C) VO 2 , (D) average VO 2 , (E) respiratory exchange ratio (RER), (F) average RER, (G) energy expenditure, (H) average energy expenditure. Data are expressed as mean + SEM. * p < 0.05, ** p < 0.01. A, C, E, G: Grey blocks denote lights off/active period. representative photomicrographs of the SCN from room air (sham) and CS-exposed mice illustrating differences in the number of Bmal-1 cells. Scale bars = 50 µm. Data are expressed as mean + SEM. ** p < 0.01, *** p < 0.001, *** p < 0.0001.

Discussion
The present study is the first to show that chronic CS exposure leads to impairments in social recognition memory and neophobia in a novel environment, and this is associated with alterations in diurnal rhythm profiles. Notably, daily administration of the antioxidant, apocynin, was effective at counteracting social recognition memory impairments and microglial area per cell but was unable to restore CS-induced neophobia, diurnal energy profiles, diurnal rhythm protein disruption or increased astrocyte density.
In the lungs, apocynin co-administration lowered CS-induced inflammation by attenuating inflammatory cell recruitment, most evidently macrophages. In line with this, the functional parameters of the lungs were significantly improved in apocynin treated mice. This is not unprecedented given that macrophage numbers have been shown to be elevated in the airways, lung parenchyma, BALF and sputum of people with COPD, which may be responsible for the initiation and perpetuation of the inflammatory response of the lungs (Akata and van Eeden, 2020). Moreover, these macrophages may be directly activated by CS exposure to secrete proteases, such as matrix metalloproteinases, which are responsible for airway remodelling and destruction of the lung parenchyma (Barnes et al., 2003). The overexuberant oxidative profile in COPD lungs, impairs alveolar macrophage-mediated efferocytosis which can be damaging as neutrophils are persistently recruited into the airways (Jubrail et al., 2017), which leads to amplified inflammation and further tissue destruction. Despite the partial reduction in the number of macrophages within the BALF following apocynin treatment, we have not assessed the activity status of the remaining macrophages (Vlahos and Bozinovski, 2014). Thus, it is possible that chronic CS exposure may interfere with the normal function of these macrophages leading to defective efferocytosis of apoptotic cells. Defective efferocytosis may lead to secondary necrosis of the uncleared cells and further adding to airway inflammation, promoting mucous metaplasia and lung destruction leading to the manifestation of chronic bronchitis and emphysema, however, further work is needed to assess macrophage function following CS exposure and whether the ROS scavenger, apocynin, is able to restore macrophage function (O'Donnell et al., 2006). representative photomicrographs of the SCN from room air (sham) and CS-exposed mice illustrating differences in astrocyte density. Scale bars = 100 µm. (E) representative photomicrographs of the SCN from room air (sham) and CS-exposed mice illustrating differences in Area Iba-1 labelling per Iba-1 positive cell. Scale bars = 50 µm. Data are expressed as mean + SEM. # interaction between exposure and treatment p < 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. Importantly, much of the disease burden and related healthcare due to COPD is associated with managing its extra-pulmonary comorbidities. Cigarette smoke-induced COPD can have significant long-term effects on neurocognition. People with COPD often experience cognitive impairments and worse mental health status (i.e., more anxious, depressed and socially deprived) compared to the non-COPD population. Importantly, worse mental health status can directly result in a lower quality of life, further cognitive decline, and more symptoms of fatigue (Franssen et al., 2018;Conti et al., 2019) which in turn will worsen the mental health status, forming a downward spiral (Hurst et al., 2020). Despite the known comorbid impacts, there is very limited literature of how COPDinduced neurocognitive impairments manifests. We have previously demonstrated that sub-chronic and chronic CS exposure can induce working memory impairments, as well as alter microglial and synaptogenesis profiles within the hippocampus De Luca et al., 2022) which was sustained beyond smoking cessation . In the present study, we have shown that lung damage induced by chronic CS exposure leads to social recognition memory impairments and neophobia to a novel environment. Interestingly, an apocynin treatment co-administered with CS exposure was able to improve this social recognition memory impairments to sham levels. Although there is very limited literature, in the preclinical setting, Xu and colleagues have shown that exposure to nicotine e-cigarettes for 43 days in mice reduced the time in the novel mouse chamber in the social interaction test compared to control mice whilst menthol flavoured nicotine e-cigarettes increased sociability (Xu et al., 2022). Exposure to waterpipe tobacco smoke in male Sprague-Dawley rats for four weeks increased anxiety-like behaviors in the elevated plus maze and open field (Hammad et al., 2022).
The causal link between anxiety-like behaviors and diurnal rhythm disruption has been elegantly reviewed (Walker et al., 2020), however, it remains uncertain whether these behavioral outcomes are through circadian control or through regulation of downstream effectors of diurnal rhythm, furthermore, the exact impact of CS exposure is still poorly understood. Several studies suggest that circadian transcriptional activator (i.e., Clock and Bmal1) and repressor (i.e., Per1/2 and Cry1/2) genes exert opposing control over neurocognitive behaviors. Depletion of the activator gene, Clock, results in hyper-locomotor activity in novel environments such as an open field arena, however, anxiety-like behaviors were comparable to wild-type mice (Easton et al., 2003;Roybal et al., 2007). Moreover, mutations in the Clock gene resulted in extreme manic-like behaviors (Arey et al., 2014). Meanwhile, SCN-specific Bmal1-knockdown in mice was sufficient to reduce the time in the light compartment during a light/dark test without impairing total locomotor activity (Landgraf et al., 2016). Conversely, depletion of the circadian clock repressor genes (Per1/2 or Cry1/2) does not affect locomotor activity but is implicated in enhanced anxiety-like behaviors (Spencer et al., 2013;Keers et al., 2012;De Bundel et al., 2013). Although there is a plethora of literature surrounding anxiety and diurnal rhythm, there is very limited knowledge in the context of social recognition memory. Moura and colleagues have shown that the phase of circadian rhythm influences social recognition memory, with an enhanced memory in the social interaction task occurring during the inactive phase (Moura et al., 2009). Moreover, disruption of the circadian rhythm in arrhythmic hamsters impairs social recognition memory (Müller and Weinert, 2016). Whilst Reijmers and colleagues showed no association between social recognition memory and circadian rhythm variations (Reijmers et al., 2001).
In our study, we show that CS-exposed vehicle mice displayed a pronounced neophobic phenotype in the object exploration task as well as an impairment in social recognition memory towards a novel mouse in the social interaction task. Apocynin prevented the social memory impairment but was unable to restore the neophobic phenotype. Alongside this, apocynin treatment did not have notable benefit in restoring the shift in diurnal energy or diurnal rhythm profiles by CS exposure. Interestingly, expression of the transcriptional repressor gene, Per2, was significancy increased in both vehicle and apocynin treated mice exposed to CS compared to sham mice. Contrary to this, CSexposed mice displayed a reduced number of Bmal1-positive cells compared to sham vehicle and apocynin mice. Thus, our data indicates that diurnal rhythm genes and proteins may influence neophobic-like behavior, but not social recognition memory, beyond its renowned role in diurnal rhythm regulation.
It has become clear that both anxiety disorders and cognitive function are associated with functional connectivity between multiple anatomical brain regions including the ventral hippocampus, medial prefrontal cortex, basolateral amygdala and the hypothalamus (Pi et al., 2020;Padilla-Coreano et al., 2016;Jimenez et al., 2018;Clewett et al., 2014). This high degree of connectivity indicates an intricate neural network that supports neurocognition in an interdependent manner. Emerging evidence from preclinical and clinical studies have highlighted the involvement of both astrocytes and microglia in neurocognition. For example, Zhou and colleagues suggest that astrocytes may play important roles in the maintenance of the excitatory-inhibitory balance and neurotrophic states of local networks that are responsible for anxiety-like behaviors (Zhou et al., 2019) and are known to regulate synaptic activity, thus, regulating cognition (Santello et al., 2019). In line with this, numerous studies have linked microglial activation with social learning and memory as well as anxiety-and depressive-like behaviors and anti-inflammatory treatments can supress both psychological behaviors and microglial profiles (Han et al., 2020;Liu et al., 2018;Wang et al., 2018;Torres et al., 2016;Komorowska-Müller et al., 2021).
Moreover, a causal link between SCN glial cells (both astrocytes and microglia) and diurnal behaviors (Tso et al., 2017;Brancaccio et al., 2019;Brancaccio et al., 2017;Sominsky et al., 2021), and the regulation of anxiety-like behaviors by SCN neurons in mice has been postulated (Vadnie et al., 2022). However, to the best of our knowledge, the relationship between SCN glial cells, diurnal regulation and social recognition memory, or the influence of environmental stressors such as CS exposure is yet to be explored. We have recently shown that chronic exposure to CS resulted in altered astrocyte and microglial profiles within the hippocampus and this is associated with improved recognition memory in the novel object recognition task De Luca et al., 2022). Similar to our findings, Hinwood and colleagues have shown that chronic stress induced working memory impairments were improved following inhibition of prefrontal cortex microglial activation (via minocycline administration) (Hinwood et al., 2013). In this study, we are the first to show that chronic CS exposure induces an increase in astrocyte and microglial density within the SCN region of the hypothalamus, which may contribute to the impairments in social recognition memory and neophobia towards a novel environment, and coadministration with the antioxidant, apocynin, was able to restore the microglial morphology but not astrocytes to sham levels.
Under normal physiological conditions, the HPA axis is involved in stress adaptation, and is tightly regulated to ensure quick and controlled responses to stressful events . Activation of the HPA axis during stress induces the secretion of corticosterone; an antiinflammatory, which coordinates the responses to the stressor and facilitates adaptive processes . Corticosterone is released in a diurnal pattern, peaking in the morning and steadily declining throughout the day (Heim et al., 2000). Although short-term stress may be adaptive, chronic stress such as CS exposure may induce maladaptive responses and hypersensitize physiological peripheral and central responses (Heim et al., 2000). Ultimately, this maladaptive stress response may perpetuate corticosterone dysfunction inducing widespread inflammation, diurnal rhythm dysfunction and behavioral changes.
To determine whether the CS exposure effects on anxiety-like behavior/social recognition memory and diurnal rhythm disruption were associated with long-term changes to the HPA axis, we measured basal circulating corticosterone following exposure to either CS or room air. The sham vehicle mice in this study displayed higher basal serum corticosterone levels compared to previous literature on BALB/c mice (approx. 3-fold increase), suggesting that chronic daily injections led to stress-induced HPA axis activation in these mice (Olfe et al., 2010). Interestingly, we demonstrate that basal serum corticosterone levels in CS vehicle mice were significantly lower than sham mice which could suggest that CS exposure may led to reduced stress-induced disturbances in the HPA axis or an exaggerated negative feedback of the HPA axis. Apocynin co-administration was able to negate the stimulatory effects of CS exposure on basal circulating corticosterone levels, suggesting inhibition of inflammation and oxidative stress may help to prevent the hyperactivation of the HPA axis. Our results are similar to clinical studies in which people with COPD have reduced salivary cortisol compared to healthy controls (Du et al., 2014;Wei et al., 2021). Another study showed that reduced serum corticosterone was associated with anxiogenic behaviors, and this reduced cortisol levels in individuals with COPD was negatively associated with a depression diagnosis (Du et al., 2014). Thus, it would be beneficial for future studies to assess whether an antioxidant treatment could restore the reduced cortisol in people with COPD and improve neurocognitive disorders in these people.
It is noteworthy that in this study, we were unable to discern whether our improved neurocognitive outcomes and microglial profiles following apocynin administration was due to the reduced pulmonary inflammation and improved lung function, or whether these improvements are independent of the lung; with apocynin directly effecting the brain or a combination of the two. Apocynin exhibits NOX inhibitory properties by blocking the assembly of a functional NOX complex, NOX2 (Johnson et al., 2002) and/or radical scavenging action (Heumu¨ller et al., 2008), depending on its concentration (Petronio et al., 2013). Numerous published studies report the ability of apocynin to penetrate the blood-brain barrier leading to improved neurological outcomes following stroke (Wang et al., 2008) and in neurodegenerative diseases via decreasing microglial activation (Ma et al., 2017). In addition, deletion of NOX2 in mice significantly reduced oxidative stress, microglial activation and improved functional outcomes following cerebral ischemia (as reviewed in (Ma et al., 2017)). Although we have demonstrated that apocynin is able to exert good anti-inflammatory actions and inhibitory effects on NOX2 beyond the pulmonary system (Oostwoud et al., 2016;Chan et al., 2021), we are unable to definitively discern whether the current neuroprotective findings are a result of this or independent via blood-brain barrier penetration. Thus, it would be beneficial for future studies to utilise specific inhibition of NOX2 activation within the CNS in a chronic CS exposure model (i.e. subcutaneous-implanted osmotic pumps containing apocynin) to assess whether improvements in social recognition memory and microglial activation are independent of pulmonary outcomes which could lead to improved therapeutic potentials.
Clinical studies have shown factors such as gender and age do not specifically contribute to the predisposition or initiation of the lung pathology in COPD directly, however, significantly influence the course of the disease (Franssen et al., 2019). Women are more likely to develop severe airflow limitation and emphysema than men; however, upon comparison between sexes with a similar degree of airflow limitation, women have better oxygenation and less emphysema but more small airway involvement and experience acute exacerbations of COPD compared to male counterparts (Celli et al., 2011). Yet, women have fewer and different comorbidities compared to males. Regarding neurocognitive disorders, Shea and colleagues reported that females with COPD had higher cognitive scores compared to males with COPD; however, overall impaired compared to healthy controls (Shea et al., 2022). A meta-analysis of 6 cohort studies revealed that patients with COPD had an increased risk of dementia, and gender did not increase the risk . However, Laurin et al. reported a nearly 56 % prevalence in females compared to 35 % in males with COPD, which may be associated with women having a higher lifetime prevalence of mood disorders than men (Laurin et al., 2007). A limitation of the present study is that our data was derived in male mice only, and previous studies have demonstrated that male and female mice respond differently to cigarette smoking (Tam et al., 2016). Hence, future studies in both male, female, and ovariectomized mice are underway to address this knowledge gap.
In summary, we showed a series of novel findings that the augmented pulmonary inflammatory profile and lung dysfunction following chronic CS exposure, is associated with social recognition memory impairments and neophobia in a novel environment and alterations in diurnal rhythm genes and proteins within the SCN of the hypothalamus. In our attempts to reverse both the pulmonary and central effects of chronic CS exposure, we also revealed that our apocynin protocol preserved social recognition memory, the serum basal corticosterone levels and microglial morphology in the SCN of the hypothalamus, but not astrocyte density, nor diurnal regulation parameters and circadian rhythm transcriptional genes and proteins. Thus, going forward, it will be imperative to evaluate other therapeutics that may have correctional effects on diurnal rhythm regulation to treat COPD-related neurocognitive impairments.