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Antidepressant effects of moxidectin, an antiparasitic drug, in a rat model of depression
Behavioural Brain Research 376 (2019) 112220
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Antidepressant effects of moxidectin, an antiparasitic drug, in a rat model of depression
T
⁎
Bruk Getachew, Yousef Tizabi
Department of Pharmacology, Howard University College of Medicine, Washington DC, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Helplessness Forced swim test BDNF TNF-alpha Hippocampus Frontal cortex Dysbiosis Gut microbiome
Substantial data indicate that an imbalance in gut microbiome (GM), also referred to as dysbiosis, may play an important role in depression. Moreover, drugs that normalize GM can result in an antidepressant-like effect. It was reported recently that moxidectin (MOX), an antiparasitic drug commonly used in veterinary medicine, has a positive influence on microbiota implicated in mood regulation. We undertook this study to determine whether MOX would actually show antidepressant-like properties in an animal model of depression and whether it would affect the hippocampal and frontal cortex levels of brain-derived neurotrophic factor (BDNF) or tumor necrosis factor (TNF)-alpha, peptides that have been implicated in pathogenesis of depression and effectiveness of various antidepressants. Adult male Wistar-Kyoto rats, a putative animal model of depression, were treated with a single dose of MOX (2.5 mg/kg, i.p.) and their performance in the open field locomotor activity (OFLA) as well as in the forced swim test (FST) was evaluated at 24 h, one week and two weeks after the single injection. A separate group of rats were injected with 2.5 mg/kg MOX and sacrificed 24 h later for neurochemical evaluations. MOX resulted in a decrease in immobility score after 24 h, whereas OFLA was not affected. Concomitant with the 24 h behavioral effects, the levels of hippocampal and frontal cortical BDNF were significantly increased, whereas the levels of TNF-alpha in both these areas were significantly decreased. The decrease in immobility scores was still evident after one week, but not 2 weeks of rest. These results indicate long lasting antidepressant effects of a single MOX dose and suggest potential utility of this drug in treatment-resistant depression.
Despondent feeling, loss of interest in pleasurable activities, guilt, worthlessness, trouble concentrating as well as abnormalities in appetite and sleep are hallmarks of major depressive disorder (MDD). MDD, which may ultimately lead to suicidal ideation and actual suicide, is a serious mental illness with relatively high prevalence (over 10% of population in US alone) [1–5]. Although the etiology remains elusive, genetic and environmental factors, particularly stress may play major roles [6–8]. The widely used antidepressants are based on the hypothesis of “Biogenic Amine Depletion” which posits that low levels of serotonin, norepinephrine, or dopamine in the brain are responsible for the symptom manifestation. However, the current “monoaminergic” drugs in addition to various side effects, have a delayed onset and limited efficacy [8,9]. Very recently esketamine nasal spray was approved as a fast-acting antidepressant in treatment-resistant patients (FDA News Release, March 5, 2019). It is believed that this drug acts primarily by inhibiting the glutamatergic NMDA receptors, although lately a role for GABA system has also been suggested [10]. Nonetheless the search for novel drugs for prevention or treatment of this crippling mental disease is continuing [11].
⁎
One of the major hinderances in novel drug development for MDD is the lack of complete understanding of its neurobiological substrates. Current hypotheses, going beyond the biogenic amine theory, postulate involvement of neurotrophic and immune (inflammatory) systems. Thus, it is believed that activation of neurotrophins such as brain-derived neurotrophic factor (BDNF) and suppression of pro-inflammatory cytokines (e.g. tumor necrosis factor alpha = TNF-α) are critical mechanisms contributing to the antidepressant effect of ketamine [11–16]. Ketamine was also shown to interact with gut microbiota, promoting probiotic bacteria and suppressing opportunistic bacteria that have a direct bearing on mood regulation [17]. Since moxidectin (MOX), an antiparasitic agent, widely used in veterinary medicine was shown to interact with the same bacteria [18], we undertook this study to determine whether MOX may also possess an antidepressant property and whether such property may be associated with enhancement of BDNF and suppression of TNF-α in the hippocampus and the frontal cortex, two brain regions intimately involved in mood regulation [19]. We used Wistar Kyoto (WKY) as these rats are extensively utilized as a putative animal model of depression [19–26]. Interestingly, these rats
Corresponding Author at: Department of Pharmacology, Howard University College of Medicine, 520W Street, NW, Washington DC 20059, USA. E-mail address: [email protected] (Y. Tizabi).
https://doi.org/10.1016/j.bbr.2019.112220 Received 8 July 2019; Received in revised form 13 August 2019; Accepted 6 September 2019 Available online 09 September 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.
Behavioural Brain Research 376 (2019) 112220
B. Getachew and Y. Tizabi
A separate group of rats were treated with 2.5 mg/kg of MOX and sacrificed by decapitation, approximately 24 h later, to coincide with the time of behavioral observation. No behavioral tests were done in these animals. This was to avoid potential confounding effects of swim test on neurochemical parameters. Brains were quickly removed, frozen on dry ice and stored at -80°C until dissection for BDNF and TNF-alpha measurement. The hippocampus (bilateral) and frontal cortex were dissected as previously described [19]. Western blot was performed as described in detail previously [19–21]. Briefly, homogenate of the dissected hippocampus was made in lysis buffer (10 mM Tris-buffer, 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100 (v/v) with protease inhibitors (Sigma-Aldrich, St. Louis, MO). The protein concentration in each sample was determined using a BCA protein Assay Kit (Pierce Biotechnology Inc., IL), and equal protein amount (as confirmed by β-actin) was loaded in each immunoblot. The proteins were separated using 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membranes were blocked with a blocking reagent (5% nonfat milk in TBS buffer) for 1⁄2 h and incubated at 4 °C overnight with the primary antibody against BDNF (1:500, Santa Cruz Biotechnology Inc., Santa Cruz, CA) or TNF-alpha (1:500, Santa Cruz Biotechnology). The membranes were washed with TBST (TBS buffer with 1% Tween-20) and blocked with the blocking reagent. Membranes were then incubated for 1 h at room temperature in Goat Anti- Rabbit-HRP conjugated secondary antibody (1:3000 in TBS, Bio-Rad Laboratories, CA). The membranes were then washed in the TBST washing solution and then visualized using enhanced chemiluminescent kits (Bio-Rad Laboratories, CA). The intensity of the protein bands on the gel was quantified using ChemiDoc XRS system (Bio-Rad Laboratories, CA). Statistical differences between treatment groups were determined by Student’s t-test as in all cases there were two treatment groups to be compared. Significant difference was set a priori at p < 0.05, two tail. We also applied the non-parametric Mann-Whitney U test analysis due to relatively small sample sizes. In all cases the significant differences indicated by the t-test were verified by Mann-Whitney U test and hence the statistical differences are indicated in terms of t values. Single treatment with MOX resulted in a significant decrease in FST immobility (approximately 62%) when tested 24 h after the injection [t = 8.64, p < 0.01]. compared to the control (Fig. 1A). Open field locomotor activity was not altered (Fig. 1B), suggesting that the treatment effects of MOX on FST were independent of any effects on general locomotion. One week after the last single injection, the effect of MOX on immobility in the FST was still evident. Hence, there was approximately 30% decrease in immobility (t = 3.25, p < 0.05) (Fig. 1A) and again no change in OFLA (Fig. 1B). After 2 weeks of rest there was still a 12% decrease in immobility score which was not statistically significant [t = 0.88, p > 0.65] (Fig. 1A). Here also, no change in OFLA was observed (Fig. 1B). Western blot analysis showed that acute treatment with the 2.5 mg/ kg/ dose of MOX resulted in increases in BDNF levels in the hippocampus (1.6-fold, p < 0.01) and the frontal cortex (1.8-fold p < 0.01) 24 h after a single administration (Fig. 2). An opposite trend was observed in terms of TNF-alpha levels in both areas. Hence, 2.5 mg/kg/ dose of MOX resulted in decreases in TNF-alpha levels in the hippocampal (2.9-fold, p < 0.01) and the frontal cortex (3.5-fold p < 0.01) 24 h after a single administration (Fig. 3). The results of the current study suggest antidepressant-like effects of an acute dose of MOX in an animal model of treatment-resistant depression. This effect was long lasting as the behavioral despair-like reflected in FST immobility scores was still down one week after MOX injection. MOX, derived from the actinomycete Streptomyces Cyanogriseus and belonging to the milbemycin family, is a third-generation macrocyclic lactone with potent insecticide activity which has been used for the treatment of internal and external parasites in cattle, sheep, deer, and horses [29–32]. In 2018, it was also approved in the
show a lower level of hippocampal BDNF and exaggerated release of pro-inflammatory cytokines in response to stressors [27,28]. Adult male WKY rats (14–15 weeks old) and weighing about 250 g, were obtained from Envigo (previously Harlan Laboratories, Indianapolis, IN). Animals receiving the same treatment were pairhoused through the duration of the experiment in a standard polypropylene shoebox cages (42 × 20.5 × 20 cm) on chip bedding. Animals were subjected to a 1-week acclimatization period upon their arrival, during which they were handled daily to minimize any handling related stress. Throughout the study, with the exception of behavioral tests, animals had free access to food (Harlan Tek Lab) and water. The room was maintained at 24–26 °C at 55–66% relative humidity, on a reverse light cycle (lights on 7:00p.m.–7:00a.m.) to allow convenient behavioral evaluations of the animals during their active period. Acclimatization to reversed dark cycle was done over a one-week period where the light hours were shifted by approximately 2 h daily. All behavioral testing and injections occurred between 8:00a.m. and 12:00 p.m. during the animal's active phase as described previously [19–22]. All experiments were carried out in accordance with NIH guidelines, and approved by the Institutional Animal Care and Use Committee of the Howard University. Animals were divided into three groups (n = 6/group) and received intraperitoneal (i.p.) injection of either saline (control) or 2.5 mg/kg dose of MOX. MOX (10 mg/kg) solution was purchased from Boehringer Ingelheim (St. Joseph, MO) and was diluted in a 0.9% sodium chloride solution (saline) to a concentration that would allow for an injection volume of 10 mL/kg of body weight at dose of 2.5 mg/kg as described previously [18]. Controls received saline. The animals from the same treatment group were housed together. The dose of MOX (2.5 mg/kg) was based on our recent in-vivo studies in rats where the effect of the same dose of MOX on gut microbiota was analyzed [18]. However, in this study we used a single injection of MOX to determine whether the antidepressant-like effect might be seen within 24 h and if so, whether the effect was long lasting. This paradigm is based on the search for fast acting and long-lasting novel antidepressants [19]. Approximately 24 h after the last injection, animals were tested in an open-field activity monitoring cage (27 × 27 × 20.3 cm, Med Associates, Inc., St. Albans, VT) for 5 min where ambulatory counts representing the number of infrared beam interruptions were recorded [19]. This behavior was assessed to determine if drug treatment affected general locomotor behavior, which might impinge on forced swim test immobility assessment [19–22]. We have consistently tested the behavioral effects of the drugs at approximately 24 h after the last injection to determine whether the desired effects are manifested within a relatively short-time period [19–22]. This time period gives sufficient time for the complete absorption and distribution of the drug and chance to manifest its pharmacodynamic effects. Immediately following the open field activity test each animal was evaluated for its behavior (immobility) in the forced swim test (FST). Briefly, each rat was placed in a Pyrex cylinder pool measuring 17 cm in diameter and 60 cm in height for 5 min. The cylinder was filled with 30 cm water (25 ± 1 °C) to ensure that the animals could not touch the bottom of the container with their hind paws or their tails. The FST activity was video recorded for subsequent analysis. The rat was removed after 5 min, dried, and placed in its home cage. A time sampling scoring technique was used whereby the predominant behavior in each 5-s period of the 300- s test was recorded. Inactivity (immobility) and activity (swimming) were distinguished as mutually exclusive behavioral states. Swimming behavior was defined as movement (usually horizontal) throughout the cylinder. Immobility was defined when no additional activity was observed other than that required to keep the rat's head above the water [19–22]. Note: Since behavioral effects were observed a day after a single MOX injection, both OFLA and FST were repeated after one week of rest and again after two weeks of rest to determine the lasting effects of the single drug injection on these parameters. 2
Behavioural Brain Research 376 (2019) 112220
B. Getachew and Y. Tizabi
Fig. 3. A depicts representative immunoblots and Fig. 3B depicts the effects of MOX on TNF-alpha levels in the hippocampus (Hipp) and the frontal cortex (FCX) of WKY rats treated with 2.5 mg/kg MOX. The animals were sacrificed 24 h after a single i.p. injection of MOX. Values are mean ± SEM. N = 6/ group. **p < 0.01 compared to control. Fig. 1. Effects of MOX on immobility scores in the forced swim test (1A) and open field locomotor activity (1B) in WKY rats. The animals were tested 24 h, then one and two weeks after a single i.p. injection of MOX (2.5 mg/kg). Values are mean ± SEM. N = 6/group. *p < 0.05, **p < 0.01 compared to control.
[34,35]. Since AUD is commonly associated with higher incidence of depression [36,37], it would be of significant clinical relevance to determine whether MOX might also be specifically effective in AUD-related depression. In this regard, we have recently reported on effectiveness of ketamine in alcohol-induced depression [11]. Ketamine has also been shown to reduce alcohol intake in some preclinical studies [38,39]. However, the use of ketamine in AUD might be problematic since ketamine might have abuse liability of its own. To our knowledge no abuse liability of MOX has been reported, hence it might offer a novel intervention in AUD and/or depression associated with AUD. Although MOX effect in AUD has been attributed to its interaction with purinergic P2 × 4 receptors [35], our results implicate a role for the neurotrophic factor, BDNF and at least one of the pro-inflammatory cytokines, TNF-alpha in antidepressant effects of MOX. This is due to the fact the levels of the BDNF in both hippocampus and the frontal cortex were elevated by MOX, whereas the levels of TNF-alpha were reduced in both these areas a day after the drug injection, concomitant with the observed antidepressant effects. Nonetheless, further understanding of MOX mechanism and potential involvement of other systems in its antidepressant effects are warranted. In addition, it is necessary to delineate potential sex differences in response to the effects of MOX as gender-dependent variation in depression and response to antidepressants is well documented [40]. In summary, the results of the current study suggest potential usefulness of MOX as a novel intervention in treatment resistant depression. Funding This work was partially supported by: NIH/NIAAAR03AA022479.
Fig. 2. A depicts representative immunoblots and Fig. 2B depicts the effects of MOX on BDNF levels in the hippocampus (Hipp) and the frontal cortex (FCX) of WKY rats treated with 2.5 mg/kg MOX. The animals were sacrificed 24 h after a single i.p. injection of MOX. Values are mean ± SEM. N = 6/group. **p < 0.01 compared to control.
Declaration of Competing Interest The authors state no conflict of interest. References
United States for onchocerciasis (river-blindness) for people over the age of 11. Interestingly, in addition to anti-parasitic properties, MOX may also have antibacterial effects [33]. Moreover, some preclinical studies have suggested its potential use in alcohol use disorders (AUD)
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Behavioural Brain Research 376 (2019) 112220
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[2] L. Perez-Caballero, S. Torres-Sanchez, C. Romero-López-Alberca, F. González-Saiz, et al., Monoaminergic system and depression, Cell Tissue Res. 377 (2019) 107–113. [3] G.R. Villas Boas, R. Boerngen de Lacerda, M.M. Paes, et al., Molecular aspects of depression: a review from neurobiology to treatment, Eur. J. Pharmacol. 851 (2019) 99–121. [4] World Federation for Mental Health, World Federation for Mental Health, (2012) www.who.int/mental_health/…/wfmh_paper_depression_wmhd_2012. [5] National Institute of Mental Health, Awareness Month: by the Numbers, (2015) www.nimh.nih.gov/…/2015/mentalhealth-awareness-month-by-the-numbers. [6] N. Lopizzo, L. Bocchio Chiavetto, N. Cattane, et al., Gene-environment interaction in major depression: focus on experience-dependent biological systems, Front. Psychiatry 6 (2015) 68. [7] M. Shadrina, E.A. Bondarenko, P.A. Slominsky, Genetics factors in major depression Disease, Front. Psychiatry 9 (2018) 334. [8] E.L. Belleau, M.T. Treadway, D.A. Pizzagalli, The impact of stress and major depressive disorder on hippocampal and medial prefrontal cortex morphology, Biol. Psychiatry 85 (2019) 443–453. [9] T.P. Blackburn, Depressive disorders: treatment failures and poor prognosis over the last 50 years, Pharmacol. Res. Perspect. 7 (3) (2019) e00472. [10] M.V. Fogaça, R.S. Duman, Cortical GABAergic dysfunction in stress and depression: new insights for therapeutic interventions, Front. Cell. Neurosci. 13 (2019) 87. [11] B. Getachew, Y. Tizabi, Ketamine and NBQX both normalize alcohol-withdrawal induced depressive-like characteristics in rats, Jour. Drug Abuse Res. 8 (2019) 236069. [12] C. Bjorkholm, L.M. Monteggia, BDNF - a key transducer of antidepressant effects, Neuropharmacology 102 (2016) 72–79. [13] T. Taniguchi, K. Shibata, K. Yamamoto, Ketamine inhibits endotoxin-induced shock in rats, Anesthesiology 95 (2001) 928–932. [14] M. De Kock, S. Loix, P. Lavand’homme, Ketamine and peripheral inflammation, CNS Neurosci. Ther. 19 (2013) 403–410. [15] E. Haroon, A.H. Miller, Inflammation effects on brain glutamate in depression: mechanistic considerations and treatment implications, Curr. Top. Behav. Neurosci. 31 (2017) 173–198. [16] W. Cui, Y. Ning, W. Hong, et al., Crosstalk between inflammation and glutamate system in depression: signaling pathway and molecular biomarkers for ketamine’s antidepressant effect, Mol. Neurobiol. 56 (2019) 3484–3500. [17] B. Getachew, J.I. Aubee, R.S. Schottenfeld, et al., Ketamine interactions with gutmicrobiota in rats: relevance to its antidepressant and anti-inflammatory properties, BMC Microbiol. 18 (2018) 222. [18] B. Getachew, R.E. Reyes, D.L. Davies, et al., Moxidectin effects on gut microbiota of Wistar-Kyoto rats: relevance to depressive-like behavior, Clin. Pharm. Exp. Med. (2018). [19] B. Getachew, L. Mendieta, A.B. Csoka, et al., Antidepressant effects of C-Terminal domain of the heavy chain of tetanus toxin in a rat model of depression, Behav. Brain Res. 370 (2019) 111968. [20] S.R. Hauser, B. Getachew, R.E. Taylor, et al., Alcohol induced depressive-like behavior is associated with a reduction in hippocampal BDNF, Pharmacol. Biochem. Behav. 100 (2011) 253–258. [21] L. Akinfiresoye, Y. Tizabi, Antidepressant effects of AMPA and ketamine combination: role of hippocampal BDNF, synapsin, and mTOR, Psychopharmacology
4
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Antidepressant effects of moxidectin, an antiparasitic drug, in a rat model of depression
T
⁎
Bruk Getachew, Yousef Tizabi
Department of Pharmacology, Howard University College of Medicine, Washington DC, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Helplessness Forced swim test BDNF TNF-alpha Hippocampus Frontal cortex Dysbiosis Gut microbiome
Substantial data indicate that an imbalance in gut microbiome (GM), also referred to as dysbiosis, may play an important role in depression. Moreover, drugs that normalize GM can result in an antidepressant-like effect. It was reported recently that moxidectin (MOX), an antiparasitic drug commonly used in veterinary medicine, has a positive influence on microbiota implicated in mood regulation. We undertook this study to determine whether MOX would actually show antidepressant-like properties in an animal model of depression and whether it would affect the hippocampal and frontal cortex levels of brain-derived neurotrophic factor (BDNF) or tumor necrosis factor (TNF)-alpha, peptides that have been implicated in pathogenesis of depression and effectiveness of various antidepressants. Adult male Wistar-Kyoto rats, a putative animal model of depression, were treated with a single dose of MOX (2.5 mg/kg, i.p.) and their performance in the open field locomotor activity (OFLA) as well as in the forced swim test (FST) was evaluated at 24 h, one week and two weeks after the single injection. A separate group of rats were injected with 2.5 mg/kg MOX and sacrificed 24 h later for neurochemical evaluations. MOX resulted in a decrease in immobility score after 24 h, whereas OFLA was not affected. Concomitant with the 24 h behavioral effects, the levels of hippocampal and frontal cortical BDNF were significantly increased, whereas the levels of TNF-alpha in both these areas were significantly decreased. The decrease in immobility scores was still evident after one week, but not 2 weeks of rest. These results indicate long lasting antidepressant effects of a single MOX dose and suggest potential utility of this drug in treatment-resistant depression.
Despondent feeling, loss of interest in pleasurable activities, guilt, worthlessness, trouble concentrating as well as abnormalities in appetite and sleep are hallmarks of major depressive disorder (MDD). MDD, which may ultimately lead to suicidal ideation and actual suicide, is a serious mental illness with relatively high prevalence (over 10% of population in US alone) [1–5]. Although the etiology remains elusive, genetic and environmental factors, particularly stress may play major roles [6–8]. The widely used antidepressants are based on the hypothesis of “Biogenic Amine Depletion” which posits that low levels of serotonin, norepinephrine, or dopamine in the brain are responsible for the symptom manifestation. However, the current “monoaminergic” drugs in addition to various side effects, have a delayed onset and limited efficacy [8,9]. Very recently esketamine nasal spray was approved as a fast-acting antidepressant in treatment-resistant patients (FDA News Release, March 5, 2019). It is believed that this drug acts primarily by inhibiting the glutamatergic NMDA receptors, although lately a role for GABA system has also been suggested [10]. Nonetheless the search for novel drugs for prevention or treatment of this crippling mental disease is continuing [11].
⁎
One of the major hinderances in novel drug development for MDD is the lack of complete understanding of its neurobiological substrates. Current hypotheses, going beyond the biogenic amine theory, postulate involvement of neurotrophic and immune (inflammatory) systems. Thus, it is believed that activation of neurotrophins such as brain-derived neurotrophic factor (BDNF) and suppression of pro-inflammatory cytokines (e.g. tumor necrosis factor alpha = TNF-α) are critical mechanisms contributing to the antidepressant effect of ketamine [11–16]. Ketamine was also shown to interact with gut microbiota, promoting probiotic bacteria and suppressing opportunistic bacteria that have a direct bearing on mood regulation [17]. Since moxidectin (MOX), an antiparasitic agent, widely used in veterinary medicine was shown to interact with the same bacteria [18], we undertook this study to determine whether MOX may also possess an antidepressant property and whether such property may be associated with enhancement of BDNF and suppression of TNF-α in the hippocampus and the frontal cortex, two brain regions intimately involved in mood regulation [19]. We used Wistar Kyoto (WKY) as these rats are extensively utilized as a putative animal model of depression [19–26]. Interestingly, these rats
Corresponding Author at: Department of Pharmacology, Howard University College of Medicine, 520W Street, NW, Washington DC 20059, USA. E-mail address: [email protected] (Y. Tizabi).
https://doi.org/10.1016/j.bbr.2019.112220 Received 8 July 2019; Received in revised form 13 August 2019; Accepted 6 September 2019 Available online 09 September 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.
Behavioural Brain Research 376 (2019) 112220
B. Getachew and Y. Tizabi
A separate group of rats were treated with 2.5 mg/kg of MOX and sacrificed by decapitation, approximately 24 h later, to coincide with the time of behavioral observation. No behavioral tests were done in these animals. This was to avoid potential confounding effects of swim test on neurochemical parameters. Brains were quickly removed, frozen on dry ice and stored at -80°C until dissection for BDNF and TNF-alpha measurement. The hippocampus (bilateral) and frontal cortex were dissected as previously described [19]. Western blot was performed as described in detail previously [19–21]. Briefly, homogenate of the dissected hippocampus was made in lysis buffer (10 mM Tris-buffer, 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100 (v/v) with protease inhibitors (Sigma-Aldrich, St. Louis, MO). The protein concentration in each sample was determined using a BCA protein Assay Kit (Pierce Biotechnology Inc., IL), and equal protein amount (as confirmed by β-actin) was loaded in each immunoblot. The proteins were separated using 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membranes were blocked with a blocking reagent (5% nonfat milk in TBS buffer) for 1⁄2 h and incubated at 4 °C overnight with the primary antibody against BDNF (1:500, Santa Cruz Biotechnology Inc., Santa Cruz, CA) or TNF-alpha (1:500, Santa Cruz Biotechnology). The membranes were washed with TBST (TBS buffer with 1% Tween-20) and blocked with the blocking reagent. Membranes were then incubated for 1 h at room temperature in Goat Anti- Rabbit-HRP conjugated secondary antibody (1:3000 in TBS, Bio-Rad Laboratories, CA). The membranes were then washed in the TBST washing solution and then visualized using enhanced chemiluminescent kits (Bio-Rad Laboratories, CA). The intensity of the protein bands on the gel was quantified using ChemiDoc XRS system (Bio-Rad Laboratories, CA). Statistical differences between treatment groups were determined by Student’s t-test as in all cases there were two treatment groups to be compared. Significant difference was set a priori at p < 0.05, two tail. We also applied the non-parametric Mann-Whitney U test analysis due to relatively small sample sizes. In all cases the significant differences indicated by the t-test were verified by Mann-Whitney U test and hence the statistical differences are indicated in terms of t values. Single treatment with MOX resulted in a significant decrease in FST immobility (approximately 62%) when tested 24 h after the injection [t = 8.64, p < 0.01]. compared to the control (Fig. 1A). Open field locomotor activity was not altered (Fig. 1B), suggesting that the treatment effects of MOX on FST were independent of any effects on general locomotion. One week after the last single injection, the effect of MOX on immobility in the FST was still evident. Hence, there was approximately 30% decrease in immobility (t = 3.25, p < 0.05) (Fig. 1A) and again no change in OFLA (Fig. 1B). After 2 weeks of rest there was still a 12% decrease in immobility score which was not statistically significant [t = 0.88, p > 0.65] (Fig. 1A). Here also, no change in OFLA was observed (Fig. 1B). Western blot analysis showed that acute treatment with the 2.5 mg/ kg/ dose of MOX resulted in increases in BDNF levels in the hippocampus (1.6-fold, p < 0.01) and the frontal cortex (1.8-fold p < 0.01) 24 h after a single administration (Fig. 2). An opposite trend was observed in terms of TNF-alpha levels in both areas. Hence, 2.5 mg/kg/ dose of MOX resulted in decreases in TNF-alpha levels in the hippocampal (2.9-fold, p < 0.01) and the frontal cortex (3.5-fold p < 0.01) 24 h after a single administration (Fig. 3). The results of the current study suggest antidepressant-like effects of an acute dose of MOX in an animal model of treatment-resistant depression. This effect was long lasting as the behavioral despair-like reflected in FST immobility scores was still down one week after MOX injection. MOX, derived from the actinomycete Streptomyces Cyanogriseus and belonging to the milbemycin family, is a third-generation macrocyclic lactone with potent insecticide activity which has been used for the treatment of internal and external parasites in cattle, sheep, deer, and horses [29–32]. In 2018, it was also approved in the
show a lower level of hippocampal BDNF and exaggerated release of pro-inflammatory cytokines in response to stressors [27,28]. Adult male WKY rats (14–15 weeks old) and weighing about 250 g, were obtained from Envigo (previously Harlan Laboratories, Indianapolis, IN). Animals receiving the same treatment were pairhoused through the duration of the experiment in a standard polypropylene shoebox cages (42 × 20.5 × 20 cm) on chip bedding. Animals were subjected to a 1-week acclimatization period upon their arrival, during which they were handled daily to minimize any handling related stress. Throughout the study, with the exception of behavioral tests, animals had free access to food (Harlan Tek Lab) and water. The room was maintained at 24–26 °C at 55–66% relative humidity, on a reverse light cycle (lights on 7:00p.m.–7:00a.m.) to allow convenient behavioral evaluations of the animals during their active period. Acclimatization to reversed dark cycle was done over a one-week period where the light hours were shifted by approximately 2 h daily. All behavioral testing and injections occurred between 8:00a.m. and 12:00 p.m. during the animal's active phase as described previously [19–22]. All experiments were carried out in accordance with NIH guidelines, and approved by the Institutional Animal Care and Use Committee of the Howard University. Animals were divided into three groups (n = 6/group) and received intraperitoneal (i.p.) injection of either saline (control) or 2.5 mg/kg dose of MOX. MOX (10 mg/kg) solution was purchased from Boehringer Ingelheim (St. Joseph, MO) and was diluted in a 0.9% sodium chloride solution (saline) to a concentration that would allow for an injection volume of 10 mL/kg of body weight at dose of 2.5 mg/kg as described previously [18]. Controls received saline. The animals from the same treatment group were housed together. The dose of MOX (2.5 mg/kg) was based on our recent in-vivo studies in rats where the effect of the same dose of MOX on gut microbiota was analyzed [18]. However, in this study we used a single injection of MOX to determine whether the antidepressant-like effect might be seen within 24 h and if so, whether the effect was long lasting. This paradigm is based on the search for fast acting and long-lasting novel antidepressants [19]. Approximately 24 h after the last injection, animals were tested in an open-field activity monitoring cage (27 × 27 × 20.3 cm, Med Associates, Inc., St. Albans, VT) for 5 min where ambulatory counts representing the number of infrared beam interruptions were recorded [19]. This behavior was assessed to determine if drug treatment affected general locomotor behavior, which might impinge on forced swim test immobility assessment [19–22]. We have consistently tested the behavioral effects of the drugs at approximately 24 h after the last injection to determine whether the desired effects are manifested within a relatively short-time period [19–22]. This time period gives sufficient time for the complete absorption and distribution of the drug and chance to manifest its pharmacodynamic effects. Immediately following the open field activity test each animal was evaluated for its behavior (immobility) in the forced swim test (FST). Briefly, each rat was placed in a Pyrex cylinder pool measuring 17 cm in diameter and 60 cm in height for 5 min. The cylinder was filled with 30 cm water (25 ± 1 °C) to ensure that the animals could not touch the bottom of the container with their hind paws or their tails. The FST activity was video recorded for subsequent analysis. The rat was removed after 5 min, dried, and placed in its home cage. A time sampling scoring technique was used whereby the predominant behavior in each 5-s period of the 300- s test was recorded. Inactivity (immobility) and activity (swimming) were distinguished as mutually exclusive behavioral states. Swimming behavior was defined as movement (usually horizontal) throughout the cylinder. Immobility was defined when no additional activity was observed other than that required to keep the rat's head above the water [19–22]. Note: Since behavioral effects were observed a day after a single MOX injection, both OFLA and FST were repeated after one week of rest and again after two weeks of rest to determine the lasting effects of the single drug injection on these parameters. 2
Behavioural Brain Research 376 (2019) 112220
B. Getachew and Y. Tizabi
Fig. 3. A depicts representative immunoblots and Fig. 3B depicts the effects of MOX on TNF-alpha levels in the hippocampus (Hipp) and the frontal cortex (FCX) of WKY rats treated with 2.5 mg/kg MOX. The animals were sacrificed 24 h after a single i.p. injection of MOX. Values are mean ± SEM. N = 6/ group. **p < 0.01 compared to control. Fig. 1. Effects of MOX on immobility scores in the forced swim test (1A) and open field locomotor activity (1B) in WKY rats. The animals were tested 24 h, then one and two weeks after a single i.p. injection of MOX (2.5 mg/kg). Values are mean ± SEM. N = 6/group. *p < 0.05, **p < 0.01 compared to control.
[34,35]. Since AUD is commonly associated with higher incidence of depression [36,37], it would be of significant clinical relevance to determine whether MOX might also be specifically effective in AUD-related depression. In this regard, we have recently reported on effectiveness of ketamine in alcohol-induced depression [11]. Ketamine has also been shown to reduce alcohol intake in some preclinical studies [38,39]. However, the use of ketamine in AUD might be problematic since ketamine might have abuse liability of its own. To our knowledge no abuse liability of MOX has been reported, hence it might offer a novel intervention in AUD and/or depression associated with AUD. Although MOX effect in AUD has been attributed to its interaction with purinergic P2 × 4 receptors [35], our results implicate a role for the neurotrophic factor, BDNF and at least one of the pro-inflammatory cytokines, TNF-alpha in antidepressant effects of MOX. This is due to the fact the levels of the BDNF in both hippocampus and the frontal cortex were elevated by MOX, whereas the levels of TNF-alpha were reduced in both these areas a day after the drug injection, concomitant with the observed antidepressant effects. Nonetheless, further understanding of MOX mechanism and potential involvement of other systems in its antidepressant effects are warranted. In addition, it is necessary to delineate potential sex differences in response to the effects of MOX as gender-dependent variation in depression and response to antidepressants is well documented [40]. In summary, the results of the current study suggest potential usefulness of MOX as a novel intervention in treatment resistant depression. Funding This work was partially supported by: NIH/NIAAAR03AA022479.
Fig. 2. A depicts representative immunoblots and Fig. 2B depicts the effects of MOX on BDNF levels in the hippocampus (Hipp) and the frontal cortex (FCX) of WKY rats treated with 2.5 mg/kg MOX. The animals were sacrificed 24 h after a single i.p. injection of MOX. Values are mean ± SEM. N = 6/group. **p < 0.01 compared to control.
Declaration of Competing Interest The authors state no conflict of interest. References
United States for onchocerciasis (river-blindness) for people over the age of 11. Interestingly, in addition to anti-parasitic properties, MOX may also have antibacterial effects [33]. Moreover, some preclinical studies have suggested its potential use in alcohol use disorders (AUD)
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