Neuroprotective effect of dexmedetomidine on autophagy in mice administered intracerebroventricular injections of Aβ25-35

doi:10.21203/rs.3.rs-2008282/v1

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Neuroprotective effect of dexmedetomidine on autophagy in mice administered intracerebroventricular injections of Aβ25-35

https://doi.org/10.21203/rs.3.rs-2008282/v1

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Current evidence suggests that dexmedetomidine (Dex) can be used as an adjuvant to general anesthesia for the elderly with or without neurodegenerative diseases, such as Alzheimer's disease (AD), since it has perioperative analgesic properties and can prevent postoperative delirium. Dysfunction involving the autophagy-lysosomal pathway is thought to underlie the pathological mechanism of AD. Evidence regarding the effects of Dex on neuronal autophagy dysfunction in mice with AD is lacking. Therefore, we hypothesized that administration of Dex could exert neuroprotective effects by ameliorating pathological autophagy dysfunction in mice that received an intracerebroventricular (i.c.v.) injection of amyloid β-protein fragment 25–35 (Aβ₂₅₋₃₅) and in an autophagy-deficient cellular model. Low dose Dex treatment reversed decreases in percentage of alternation in the Y-maze test. It restored levels of phosphorylated Ca²⁺/calmodulin-dependent protein kinase II (p-CaMKII) and postsynaptic density-95 (PSD-95), both memory-related proteins. Dex also protected synapses from Aβ-induced toxicity in mice injected with Aβ₂₅₋₃₅. Furthermore, increased expression of the autophagy-related microtubule-associated protein light chain3- II (LC3-II), p62, and lysosome-associated membrane protein2 (LAMP2) in Aβ₂₅₋₃₅ mice was reduced after low-dose Dex treatment, ameliorating aberrant autophagic reflux. The present study demonstrated that low-dose Dex treatment ameliorated memory and learning impairments. It’s neuroprotective mechanism was associated with autophagic flux in a murine Aβ₂₅₋₃₅ model. These findings suggest that Dex could represent an effective clinical approach for AD patients as a neuroprotective adjuvant in anesthesia.

Dexmedetomidine

Autophagy

Alzheimer's disease

Amyloid β-protein

Dexmedetomidine (Dex) is a highly selective α₂ adrenergic receptor agonist that is clinically used to induce perioperative analgesia with opioid-sparing agents or for sedation of patients in intensive care units [1]. Dex has generated great interest because it does not inhibit respiration or cause severe hemodynamic changes, unlike benzodiazepine, propofol, and opioids, and acts on sleep pathways at the locus coeruleus, independent of N-methyl-D-aspartate (NMDA) or gamma-aminobutyric acid A (GABA_A) receptors [2].

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases linked to progressive loss of synapses in the cerebral cortex and hippocampus, resulting in cognitive decline [3]. The pathological hallmarks of AD include extracellular accumulation of amyloid-beta (Aβ) protein and abnormally processed intraneuronal tau protein, also called neurofibrillary tangles (NFTs), which contribute to the development of AD via inducing autophagy-lysosomal pathway dysfunction [4].

Autophagy is the degradation of unnecessary cytoplasmic substrates via autophagosomes [5]. During initiation of autophagosome formation, a microtubule-associated protein light chain 3 (LC3), and its lipidated form LC3-II, and p62, induce proteasomal degradation of ubiquitinated proteins [6], and are considered key factors [7]. P62 plays a role in the fusion of lysosomes with autophagosomes to form autolysosomes; lysosome-associated membrane protein 2 (LAMP2) is an important factor in this process, which prevents accumulation of autophagosomes [8]. Subsequently, unnecessary proteins, such as LC3-II and p62 [9], are degraded by the lysosomal protease cathepsin D [10]. This process is termed autophagic flux. AD is a disease condition attributed to impaired autophagosome clearance, which may be responsible for generating Aβ protein [11, 12].

Previous studies have focused on the neuroprotective effects of Dex using different animal models, such as cerebral ischemia models [13], hypoxic models [14], oxygen-glucose-deprivation-induced injury models [15], and inflammatory models [16]. Others have demonstrated the protective properties of Dex against neurotoxicity induced by inhaled anesthetic agents, such as isoflurane [17, 18] and sevoflurane [19–23]. However, there is a lack of evidence regarding the neuronal effects of Dex on autophagy dysfunction in mice with AD.

We hypothesized that Dex might have neuroprotective effects on pathological autophagy dysfunction in AD. Therefore, we conducted behavioral tests using mice given intracerebroventricular (i.c.v.) Aβ₂₅₋₃₅ injections and analyzed memory- and autophagy-related proteins in the hippocampus to elucidate the neuroprotective effects of Dex in the murine Aβ₂₅₋₃₅ model both in vivo and in vitro.

Compliance with Ethical Standards

This animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University (Approval No. EWHA MEDIACUC 21-010-t).

Experimental animals

A total of 137 C57BL/6 mice (weight, 25 ± 3 g; age, 8-10 weeks) were purchased from DBL Co., Ltd. (Umsung-gun, Korea). Animals were kept in plastic cages and given ad libitum access to water and food under a 12-h light-dark cycle (lights on from 07:00 to 19:00). The cages were individually ventilated and located in a specific pathogen-free graded facility.

Aβ_25–35intracerebroventricular injection

Aβ_25–35 injection was performed as previously described [24,25]. Briefly, mice were anesthetized using isoflurane (1.6 - 2%) (Hana Pharm Co., Seoul, Korea) and placed in a stereotaxic apparatus. A Hamilton syringe attached to a Nanomite Injector Syringe Pump (Harvard Apparatus, Holliston, MA, USA) was used for i.c.v. injection of Aβ_25-35 (10 nM/3 μl per mouse) or saline (3 μl) at a rate of 1 µl/min, using the following coordinates: anteroposterior, 0.3 mm from bregma, mediolateral, 1.0 mm from the midline, and depth, 2.5 mm from the skull. The needle was removed 3 min after i.c.v. injection and the incision site was sutured.

Experimental preparation procedure

To determine the Dex (Sigma-Aldrich, Darmstadt, Germany) dose regimen, we conducted a preliminary study using previous hypoxia resistance model [14]. The supra-clinical dose of Dex was determined using a neuroapoptosis model [26] to determine the anesthetic dosage regimen. Loss of righting reflex response (LORR) was used to estimate the response after the mice were placed in the supine position [20]. LORR grading ranged from 0 to 4: 0 = no response, 1 = delayed attempt to right itself, but failing, 2 = delayed, uncoordinated return to the upright position, 3 = sedated, but righting themselves in a coordinated fashion, or 4 = awake, not remaining supine. At an intraperitoneal (IP) dose of 40 µg/kg (identified from preliminary data), Dex sedated mice for approximately 30 min with a LORR grade of 2 or 3. On the other hand, mice were hypnotic, with an LORR grade of 0 or 1, when treated with 80 µg/kg of Dex. The anesthetic dosage regimen was finally decided on as follows: 40 µg/kg was considered as a sedative dose which produced a LORR score grade of 2 or 3, whereas 80 µg/kg was considered as a hypnotic dose which resulted in a LORR score grade of 0 or 1. The mice were reinjected with the same quantity after 30 min to maintain sedation or hypnosis for approximately one hour. After administration of Dex, mice were placed in a rodent intensive care unit and monitored using a digital thermometer (Testo 925, Testo Limited; Hampshire, UK) and maintained at 36.5–37.5°C using a heating pad (TP472, Gaymar Industries Inc; Orchard Park, NY, USA) until they awoke. A total of 137 mice were allocated to six groups according to the type of drug: saline i.c.v. + saline IP (control, n = 23); saline i.c.v. + Dex 40 µg/kg IP (D40, n = 21); saline i.c.v. + Dex 80 µg/kg IP (D80, n = 24); Aβ_25-35 i.c.v. + saline IP (Aβ, n = 25); Aβ_25-35 i.c.v. + Dex 40 µg/kg IP (Aβ/D40, n = 23); and Aβ_25-35 i.c.v. + Dex 80 µg/kg IP (Aβ/D80, n = 21).

Y-maze test

The Y-maze test, which estimates short-term memory in mice [27], was conducted in the afternoon on days 1, 3, and 7 after IP injection. The testing maze consisted of three black arms (30 × 6 × 15 cm) oriented at a 120° angle, which were designated A, B, and C, respectively. Each mouse performed the test for 10 min on days 1, 3, and 7 after IP injection. We measured the number of consecutive arm entries, and spontaneous alternations were counted when the mice explored previously unvisited arms. For example, AAB or BCB did not count, and only three different arm entries, such as ABC or CAB, were counted as alternations. Spontaneous alternation behavior was calculated as the percentage of alternations versus total alternation opportunities (total arm entries minus two) [28]

Protein extraction and western blot analyses

Following behavioral tests, the mice were decapitated; the tissue dissected from the left hippocampus was lysed in ice-cold lysis buffer (50 mM Tris–HCl, 1% IGEPAL, 0.25% deoxycholic acid, 150 mM NaCl, 1 mM ethylene glycol tetra acetic acid, and 1 mM NaF) with a protease inhibitor (Roche Diagnostics GmbH, Penzberg, Germany) followed by sonication. Memory- and autophagy-related proteins (PSD-95, p-CaMKII, LC3-II, p62, LAMP2) were analyzed by western blotting. Protein concentrations were determined using the Bradford assay, and equal amounts of protein from each sample were analyzed. Each sample was boiled for 5 min and 50 mg/ml protein was subjected to SDS-PAGE on 4%–15% precast gels. The proteins were transferred onto blotting membranes (0.2 μm nitrocellulose membrane, Bio-Rad Laboratories, Inc., CA, USA) and blocked for 1 h with Tris-buffered saline and Tween (TBST; 20 nM Tris, 500 nM NaCl, and 0.05% Tween 20) containing 10% skim milk. The blotting membrane was probed overnight at 4ºC with primary antibodies (Supplemental Table.1), followed by incubation for 2 h with secondary antibodies (Supplemental Table 2). Actin was used as a loading control. Immunoreactive bands were visualized with a luminescent image analyzer (ImageQuant LAS 4000 mini, GE Healthcare, Buckinghamshire, UK), using a chemiluminescence reagent (Amersham ECL Prime, GE Healthcare; Buckinghamshire, UK). Densitometric analysis of the bands was performed using the ImageJ software v.1.8.0 program from the National Institutes of Health.

Immunofluorescence staining

Right hemisphere tissues (bregma: -1.3 to -2.5 mm) were fixed for 48 h in 0.1 M phosphate-buffered (pH 7.4) 4% paraformaldehyde (Merck; Darmstadt, Germany) and stored at 4ºC in a 30% sucrose solution. A freezing microtome (Leica CM 1850; Wetzlar, Germany) was used to make 30 μm cryosections of hippocampal tissue. Cryosections were incubated for 1 h in a blocking solution containing 0.2% Triton X-100 and 10% bovine serum albumin in phosphate-buffered saline (PBS). The section were incubated overnight in primary antibodies, followed by secondary antibody incubation for 2 h (SI.1, SI.2.). Brain tissues were mounted with mounting medium (VECTASHEILD, Antifade mounting medium with DAPI, Vector Laboratories, Burlingame, CA, USA) and images were obtained using a confocal microscope (ZEISS LSM 800, Zeiss; Jena, Germany).

Organotypic hippocampal slice culture (OHSC) and drug treatments

Brains of the 8-day-old Sprague-Dawley rats were immersed in an ice-cold dissecting medium. Transverse slices of hippocampi (400 μm), made with a McIlwain tissue chopper (Mickle Laboratory Engineering; Surrey, UK), were placed on semi-porous Millicell membranes in plastic inserts (0.4 μm, Millicell-CM, Millipore; Burlington, MA, USA). The membranes were then transferred to a 5% CO₂ incubator at 37°C in 6-well culture plates (Falcon, Becton Dickinson; Franklin Lakes, NJ, USA) containing neurobasal culture medium (Gibco BRL; Grand Island, NY, USA). The media was changed twice weekly for 3 weeks until the experiments ended. Aβ_25–35 (Sigma-Aldrich; St. Louis, MO, USA) was prepared as a 1 mM stock solution in sterile deionized water, stored in small aliquots at -70°C, and then incubated at 37°C for 24 h to obtain the aggregated form. Then, hippocampal slices were grown in media supplemented with 2.5 μM Aβ_25–35 for 3 days, as per our previous study. This concentration induced apoptosis but not prominent cell death [29]. The autophagic deficit model of OHSCs induced with Aβ_25–35, which is related to the deficit of autophagic flux, was created according to our previous study [30]. Hippocampal slices were incubated with 0.5, 1, 2.5, and 5 μM Dex along with Aβ_25–35 according to the experimental protocol of a previous study [13].

Propidium iodide (PI) uptake and western blot analysis of OHSCs

The extent of neuronal cell death was assessed using the fluorescent exclusion dye propidium iodide (PI). PI was added (2 μg/ml) to the culture medium and incubated in the dark for 1 h at 37°C, followed by washing with PBS, and the presence of PI was estimated by total final fluorescence at the end of each experiment. Images were obtained using an inverted fluorescence microscope (Zeiss Axiovert 200, Zeiss, Göttingen, Germany), and the image intensity was analyzed using the Axio Vision v.4.7.1.0 program (Imaging Solutions GmbH, Munich, Germany). To confirm the effect of Dex (0,1, and 2.5 μM) on the Aβ_25–35 solution, we analyzed p-tau protein, which is known as a pathologic hallmark in AD [31], and other memory- and autophagy-related proteins (PSD-95, p-CaMKII, LC3-II, p62, LAMP2) by western blotting.

Statistical analysis

Results are presented as means ± standard error of mean (SEM). The data were analyzed using one-way analysis of variance (ANOVA) using StatView v.5 software (SAS Institute, Inc., Cary, NC, USA). Fisher’s post-hoc test was used to determine means that were significantly different from the control mean. Statistical significance was set at P < 0.05.

Y-maze test

In the Y-maze test, we found a significant decrease in the percentage of alternation in the Aβ group compared to that in the control on days 1 and 7 (n = 6, P = 0.010 and 0.017, respectively; Fig. 1), and the percentage of alternation in the Aβ/D40 group recovered almost to the level of the control on days 3 and 7. In addition, percentage of alternation in the Aβ/D40 group significantly increased compared to that of the Aβ group on day 7 (n = 9, P = 0.020; Fig. 1).

Low-dose Dex decreased p-tau in Aβ_25-35 injected mice

A significant increase in the intensity of p-tau was observed in the Aβ group compared to that in the control on days 1, 3, and 7 (P = 0.004, < 0.001, and 0.009, respectively; Fig. 2). Aβ/D40 mice exhibited significantly reduced intensity of p-tau compared to that in the Aβ group on days 1, 3, and 7 (P = 0.011, 0.044, and 0.027, respectively; Fig. 2). From microscopic images, p-tau levels were increased in the pyramidal cell layer in the Aβ group compared to those in the control (Fig. 3p), and a significant decrease was observed following Dex administration (40 μg/kg) on day 1 (Fig. 3t).

Low-dose Dex attenuated dysregulation of PSD-95 and p-CaMKII in Aβ_25-35 injected mice

PSD-95 and p-CaMKII levels in the Aβ group significantly decreased and increased, respectively, compared to that in control animals on days 1, 3, and 7 (P < 0.001, < 0.001, and < 0.001, P = 0.005, 0.028, and 0.001, respectively; Fig. 4a, b). Aβ/D40 mice exhibited a significant increase and decrease in the intensity of PSD-95 and p-CaMKII, respectively, compared to that of the Aβ group on days 1, 3, and 7 and days 1 and 7 (P = 0.014, 0.004, and 0.037, P = 0.006 and 0.001 respectively; Fig. 4a, b). However, there was no significant difference in PSD-95 and p-CaMKII abundance among the i.c.v. saline groups (Fig. 4a, b).

Low-dose Dex attenuated the increase of LC3-II, p62, and LAMP2 levels in Aβ_25-35 injected mice

There were significant increases in the levels of LC3-II and p62 in the Aβ group compared to that in the control on days 1, 3, and 7 (P = 0.038, 0.011, and 0.011, respectively; Fig. 5a, P = 0.003, 0.020, and 0.037, respectively; Fig. 5b). In the Aβ/D40 group, the expression levels of LC3-II and p62 were significantly reduced compared to that in the Aβ group on day 7 (P = 0.045; Fig. 5a, P = 0.006; Fig. 5b). However, the intensities of LC3-II staining on day 3 and p62 levels on day 1 in the D80 group significantly increased compared to that in the control group (P = 0.016; Fig. 5a, P = 0.022; Fig. 5b), and were almost equal to the levels in the Aβ group.

LAMP2 levels significantly increase in the Aβ group compared to that in the control group on days 1, 3, and 7 (P = 0.018, 0.041, and 0.016, respectively; Fig. 5b), but significantly decreased in the Aβ/D40 group compared to that in the Aβ group on days 1, 3, and 7 (P = 0.033, 0.041, and 0.027, respectively; Fig. 5c). However, there were no significant differences among the i.c.v. saline groups regardless of Dex treatment. Moreover, confocal microscopic images demonstrated that there were significant increases in the co-localized immunoreactivity of LC3 and p62 in the Aβ group (Fig. 6p), and significant dissociation of LC3 and p62 was demonstrated in the Aβ/D40 group (Fig. 6t) on day 7.

Dex attenuated Aβ_25-35-induced autophagy in OHSCs

PI uptake was increased in OHSCs cultured with Aβ_25–35 compared to that in the control, which indicated an increase in neuronal cell death. Administration of Dex at four different concentrations (0.5, 1, 2.5, and 5 μM) to Aβ_25–35-treated OHSCs resulted in significantly decreased uptake of PI in the pyramidal cell layer compared to that in Aβ_25–35-only treated OHSCs (n = 6, P = 0.002, < 0.001, < 0.001, and < 0.001, respectively, Fig. 7a, b). This suggested that Dex treatment reduced neuronal cell death in OHSCs.

Low-dose Dex attenuated Aβ_25-35-induced production of p-tau and PSD-95 and p-CaMKII dysregulation in OHSCs

A significant increase in the abundance of p-tau was observed in the Aβ_25–35-only treated media, as well as in the 2.5 μM Dex-only treated media (each n = 9, P < 0.001, and 0.021, respectively; Fig. 8a). Administering 1 μM Dex to the Aβ_25–35-supplemented media attenuated the p-tau production compared to the that in Aβ_25–35-only treated media (each n = 9, P = 0.001; Fig. 8a). PSD-95and p-CaMKII significantly decreased and increased, respectively, in OHSCs treated with only Aβ_25–35 compared to that in the control (n = 9, P < 0.001 and 0.004, respectively; Fig. 8b, c). The intensity of PSD-95 signal increased and that of p-CaMKII significantly decreased in cultures treated with μM Dex 1 and Aβ_25–35 compared to that in cultures treated with only Aβ_25–35(n = 9, P = 0.011 and 0.015, respectively; Fig. 8b, c). The solution treated with only 2.5 μM Dex exhibited a significant decrease in PSD-95 and a significant increase in p-CaMKII levels compared to that in the control (n = 9, P = 0.008 and < 0.001, respectively; Fig. 8b, c).

Low-dose Dex attenuated increased LC3-II, p62, and LAMP2 levels in OHSCs treated with Aβ_25-35

The autophagy-related proteins, LC3-II, p62, and LAMP2 were significantly upregulated in OHSCs treated with only Aβ_25–35 compared to that in the control (n = 6, P = 0.023, 0.004, and 0.004, respectively; Fig. 8d-f). The intensity of LC3-II, p62, and LAMP2 staining significantly decreased after treatment with 1 μM Dex and Aβ_25–35 compared to that produced by Aβ_25–35 treatment (n = 6, P = 0.031, 0.010, and 0.018, respectively, Fig. 8d-f). However, there was no significant difference between the control and Dex (1 μM or 2.5 μM)-only treated OHSCs.

The major finding in the present study is that low-dose Dex is neuroprotective and amliorates memory and cognitive impairment induced by Aβ_25-35 via regulation of autophagic flux. The recovered percentage in the Y-maze test after treatment with low-dose Dex revealed improvements in memory and cognitive impairments, as well as, treatment with low-dose Dex resulted in a decrease and increase in the activity of the memory-related proteins p-CaMKII and PSD-95, respectively. The present study demonstrated for the first time that increased LC3-II, p62, and LAMP2 levels under Aβ_25-35 treatment were reversed after low-dose Dex administration both in vivo and in vitro, indicating improvement in otherwise aberrant autophagic reflux under Aβ_25-35 conditions.

Aβ accumulation underlies the impaired autophagic flux in AD pathogenesis [32]. Aβ_25-35, an easily synthesized active form of Aβ [33], has the same effect as the full-length Aβ protein and has been used to determine autophagy-related mechanisms involved in development of AD [34]. Based on previous studies, we established an Aβ-induced autophagy model using Aβ_25-35 to identify the neuroprotective effects of Dex. Increased immunofluorescence of p-tau proteins after i.c.v administration of Aβ_25-35 revealed the proper Aβ_25-35 animal model.

Consistent with the decreased percentage of alternation seen in the Y-maze results in the Aβ group, the levels of memory-related proteins p-CaMKII and PSD-95 increased and decreased in the Aβ group, respectively; this was reversed in the Aβ/D40 group. PSD-95 is a scaffold protein that enhances synaptic transmission in the cerebral cortex [35]; low levels of PSD-95 are associated with vulnerability of synapses to Aβ [36]. Our study findings showed that there was a significant increase in PSD-95 abundance in the Aβ/D40 group on days 1, 3, and 7, providing evidence that increased PSD-95 protects synapses from Aβ toxicity. CaMKII plays an essential role in synaptic plasticity and spatial memory formation [37]. Furthermore, redistributed T286-autophosphorylation of CaMKII contributes to cognitive impairment via damaged hippocampal synapses in AD, resulting in neuronal cell death [38]. Gardoni et al. [39] suggested that reduced expression of PSD-95 in neurons is related to neuronal vulnerability mediated by direct activation of the CaMKIIα transduction pathway, which is consistent with our findings.

Our study also showed Aβ-induced upregulation of the autophagy-related proteins LC3-II, p62, and LAMP2 in Aβ_25-35mice was ameliorated by low-dose Dex treatment. A decrease in p62 is often related to accelerated autophagic flux [40], as it is as a marker for autophagic cargo degradation in autolysosomes [41]. The levels of lysosomal membrane protein LAMP2, an autolysosomal marker useful for predicting responses to interventions [42], increased along with levels of LC3-II and p62 in the Aβ group. This result demonstrated the activation of autophagic induction; it also indicated impairment in formation of single-membraned autolysosomes, thereby indicating impaired autophagic flux, in vivo and in vitro in the Aβ group. Furthermore, co-localized immunoreactivity of LC3-p62 was demonstrated in the Aβ group, which was dissociated following 40 μg/kg Dex treatment, proving that the enhancement of autophagic flux in which double-membraned autophagosomes were transformed to single-membraned autolysosomes was secondary to successful autolysosome formation.

However, we did observe elevated levels of LC3-II, p62, and LAMP2 in the D80 group compared to that in the control, and there was no significant decrease in the Aβ/D80 cohort. Previous studies have demonstrated that higher doses of Dex do not enhance its neuroprotective properties [14,26,43]. In this study, results from the Aβ/D80 group, which indicated no significant attenuation of impaired autophagic flux, could be attributed to hemodynamic side effects (e.g., bradycardia and hypotension) of high-dose Dex.

Since autophagy is a complex mechanism in terms of the degradation of abnormal proteins such as Aβ and hyperphosphorylated tau, aberrant autophagy leads to aggravation of Aβ clearance [44], and insufficient autophagy attenuates Aβ secretion in AD [45]. Therefore, researchers have targeted autophagy-related signaling pathways involving Aβ in AD therapy [41], even though a specific autophagic pathogenesis has not yet been elucidated. Consistent with our study, Shin et al. [46] demonstrated that the accumulation of autophagosome-related proteins is related to impaired autolysosome formation in Aβ-treated cell and animal models. Zhong et al. [47] also reported similar results, demonstrating that Orientin treatment reduced the elevated levels of LC3-II, p62, and cathepsin D in transgenic AD mice owing to decreased accumulation of autophagosomes due to enhanced autophagy flux. Consistent with previous studies, the present study demonstrated that Dex ameliorated memory and cognitive impairment in mice injected with Aβ_25-35 by enhancing autophagic flux. To the best of our knowledge, no study has demonstrated the neuroprotective effect of Dex, both in vivo and in vitro, via an autophagic mechanism in AD models. Our study findings suggest a mechanism underlying the neuroprotective properties of Dex and provide a basis for using Dex as an anesthetic adjuvant in patients with neurodegenerative diseases, particularly AD.

The present study had certain limitations. First, the plasma concentration of Dex was not measured in the in vivo experiments; nonetheless, we observed the LORR responses of mice treated with sedative or hypnotic doses for an hour in the preliminary study. The plasma concentration at which significant, yet arousable, sedation was achieved was approximately 0.2–0.3 ng/ml, and Ebert et al. [48] demonstrated that unarousable deep sedation occurred at plasma concentrations > 1.9 ng/ml. With a molecular weight of 236.74 g, these Dex concentrations are equivalent to 0.84–8.03 μM (molar concentration [mol/l] = mass concentration [g/l]/molecular weight). Second, it was difficult to administer Dex intravenously tomice, and we administered Dex twice IP at 30-min intervals. We decided on sedative or hypnotic doses and IP intervals of Dex by LORR scores via preliminary experiments to overcome this limitation. Third, we assessed physiological changes in mice during Dex IP injections instead of measuring vital signs such as heart rate, blood pressure, and peripheral oxygen saturation. Nevertheless, no cases exhibited fatal physiological changes, such as cyanosis, respiratory distress, and hypothermia. However, further studies would be required to measure vital signs to evaluate potential hemodynamic side effects of Dex.

In conclusion, the present study demonstrated that low-dose Dex could attenuate memory and learning impairments in Aβ_25-35i.c.v. injected mice via signaling pathways regulated by p-CaMKII and PSD-95. We confirmed the neuroprotective mechanism of low-dose Dex, which is linked to its alleviation of impaired autophagic flux. These findings suggest that Dex could be clinically used as a neuroprotective adjuvant in anesthesia for patients with AD.

AD: Alzheimer’s disease

Aβ_25–35: amyloid β-protein fragment 25–35Dex: Dexmedetomidine

i.c.v.: intracerebroventricular

IP: intraperitoneally

NFTs: neurofibrillary tangles

LC3: microtubule-associated protein light chain 3

LAMP2: lysosome-associated membrane protein 2

Nrf2: nuclear factor erythroid 2 – related factor

p-CaMKII: phosphorylated Ca2+ /calmodulin-dependent protein kinase II

PSD-95: postsynaptic density-95

OHSC: organotypic hippocampal slice culture

LORR: loss of righting reflex

PBS: phosphate-buffered saline

PI: propidium iodide

SEM: standard error of the mean

Ethics approval

This animal study was conducted according to the criteria approved by the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University (Approval No. EWHA MEDIACUC 21-010-t).

Consent to participate

Not applicable

Consent to publication

Not applicable

Availability of data and materials

Additional data can be found in the Supplementary materials. Data are available upon reasonable request.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Preprint sharing

Pre- print could be accessed at https://dspace.ewha.ac.kr/handle/2015.oak/260072?mode=full.

Author Contributions

All authors contributed to the study conception and design.

Youn Young Lee M.D., Ph.D. This author helped design the study; conduct the study; collect, analyze and interpret the data; review the literature; and write the manuscript.

Jong In Han M.D., Ph.D. This author helped design the study; review the literature; critically revise the manuscript; and approve the final version of the manuscript. This author also actively contributed to the original research project.

Sooyoung Cho, MD, PhD^.This author helped design the study; review the literature; critically revise the manuscript; and actively contributed to the original research project.

Eun Cheng Suh, M.Sc. This author helped conduct the study; collect and analyze the data; review the literature; and write the manuscript. This author also actively contributed to the original research project.

Kyung Eun Lee, M.D., Ph.D. This author helped to design the study; collect, analyze and interpret the data; was a scientific consultant; reviewed the literature; critically revised the manuscript; and approved the final version of the manuscript. This author also actively contributed to the original research project.

Acknowledgements

The authors would like to thank the entire team of the College of Medicine, Ewha Womans University, Pharmacology Laboratory.

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Neuroprotective effect of dexmedetomidine on autophagy in mice administered intracerebroventricular injections of Aβ25-35

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