Intraventricular T3 Reverses Chronic Restraint Stress-Induced Depressive-Like Behaviors: Inhibition of NF-κB/ NLRP3 Inammasome Pathway in the Hippocampus

In this study, the effects of triiodothyronine (T3) were evaluated on the NLRP3 inammasome complex formation in the hippocampus of the rat with restraint stress-induced depressive-like behaviors.Thirty-six Wistar male rats were randomly allocated to the following groups: Control, Model, and Model + T3. In Model or Model + T3 group, a single dose of PBS or T3 () was administered into the lateral ventricle. Depressive-like behaviors were induced by chronic restraint stress. The forced swimming (FST), tail suspension (TST), and open eld (OFT) tests were used to investigate depression. The rats were sacriced, and brain tissues were stored for molecular and pathological evaluations. Chronic stress increased the immobility of rats in the Model group according to FST, TST, and OFT (P < 0.05). T3 signicantly improved depressive-like behaviors (P < 0.05). The gene expression and protein level of hippocampal NF-κB, NLRP3, ASC, and Caspase-1 signicantly increased in the Model group compared to the control group (P < 0.05). The reduced hippocampal levels of NF-κB, NLRP3, ASC, and Caspase-1 were seen in the T3 group compared to the Model group (P < 0.05). Also, the Nissl staining of the CA1 region showed an increased number of dark neurons (P < 0.05) and reduced pyramidal layer thickness (P < 0.05) in the Model group. These histopathological alterations were changed by T3 administration compared to the Model group (P < 0.05). The ndings of conrmed the therapeutic effects of intraventricularly T3 on depressive-like behaviors induced by restraint stress via surviving pyramidal neurons of the CA1 region and inhibition of NF-κB/NLRP3 inammasome pathway.


Introduction
The function of several organs and systems, especially the central nervous system (CNS) and the endocrine system, can be affected by unpredictable and undesirable stressful conditions. Chronic and long-term stress contributes to the induction of a wide range of mental disorders, e.g., anxiety and depression [1]. Depression is an alteration in cognitive behavior and mood, featured by cognitive dysfunction, pleasure loss, negative mood, social isolation fatigue, sleep disorders, appetite loss, and other metabolic changes [2,3]. Among different regions of brain, it has been shown that reduced volume of hippocampus is strongly correlated with the severity of depression [4] Long-term unpredicted and repeated stress triggers a cascade of in ammatory responses in the brain [5].
There is a growing body of evidence from preclinical and clinical studies indicate that depression is attributed to the higher levels of cytokines, such as interleukin (IL)-6, IL-1β in blood and CSF [6]. The elevated levels of in ammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), IL-1β, and IL-6 in brain regions, including the hypothalamus, hippocampus, or prefrontal cortex, have been reported following depression [7,4]. The nucleotide-binding domain, leucine-rich repeat (NLR) pyrin domain protein 3 (NLRP3) in ammasomes, an intracellular multi-protein complex, is activated in various neurological and psychological disorders [8]. Full assembly and activation of the NLRP3 complex depend on two important factors of nuclear factor kappa B (NF-κB) signaling pathways and interaction of NLRP3 protein with the rest of the in ammasome machinery, including apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase 1 [9]. After activation of complex, procaspase 1 is cleaved into caspase 1, which activates IL-1β from the pro-IL-1β [10]. A growing body of evidence con rmed that NF-κB is an important mediator of stress-induced neurological impairments [11].
Up to now, several animal models have been utilized to investigate the pathological events of the depression progression or effects of antidepressants. The models with exposure of the animal to stressors with their responses, which mimic human depression, are the most popular models [12]. Chronic restraint stress is a low-cost stressor that induces a series of behavioral changes similar to those seen in humans [13].
Different classes of antidepressants have been developed based on the hypothesis that depression, as a neuroendocrine disorder, is resulted from an imbalance in the monoamine neurotransmitters [14].
However, several side effects, e.g., sexual dysfunction, cardiovascular disease, and osteoporosis, have been reported for these antidepressants [15,16]. Recently, the relationship between thyroid axis abnormalities and psychiatric disorders has been proven [17]. Triiodothyronine (T3) and thyroxine (T4), two major thyroid hormones (THs), are secreted from the thyroid gland [18]. Both hyper and hypothyroidism patients experience numerous neuropsychiatric manifestations, such as anxiety and e depressive disorders [19]. Additionally, a "low T3 syndrome", decreased peripheral conversion of T4 to T3 in the normal production of TH, has been described in patients with psychiatric depression [20]. THs treatment of patients with mood disorders, particularly depressive disorders, was reported in numerous studies [21]. It was also demonstrated that T3 could augment and accelerate treatment response with antidepressants [22]. T3 and T4 can cross the blood-brain barrier (BBB) via monocarboxylate 8 (MCT8), a speci c transporter of THs [23]. The effects of T3 are mediated by its binding to TH receptor (TR) isoforms, predominantly TRα1 and TRβ1. Notably, TRα1 is located on nearly all types of neurons [24].
Thus, the adequate local concentration of T3 is important for TRs activation [25]. T3 (the active and less TH) contributes to numerous biologic functions in normal and pathologic conditions via different mechanisms. THs regulate the expression of oxidative stress products, glutamate, and neurotrophic factors in neurological disorders [26]. Furthermore, the anti-in ammatory effects of THs were reported in several studies [27,28].
Due to the bene cial effects of T3, we used a single dose of intraventricular (IVC) T3 to evaluate its antidepressive effects in a chronic restraint stress model. To understand the mechanisms of effects of T3 as a therapeutic approach, we designed an experiment to examine: (1) depressive-like behaviors of animals, In the present study, 36 male Wistar rats (weighing 150-220 g) were kept in the standard temperature (23+1°C), 12/12 h light/ dark cycle with ad libitum access to food and water.

Experimental design
The study protocol was designed as demonstrated in Figure 1. Rats were randomly allocated to three groups (n = 12 in each group): Control (healthy animals), Model: with rats subjected to restraint stress and IVC injected with phosphate-buffered saline (PBS) [29], Model+T3: rats subjected to restraint stress and injected IP with T3. The rats in the Model and Model+T3 groups were exposed to stress. For this purpose, each rat was placed in a plastic cylinder (3 h/day from 8:00 am to 11:00 am) for 14 consecutive days [29]. Twenty-four hours after model induction, the animals in the Model+T3 group were injected with IVC T3 (a single dose of 25 ug/kg body). T3 was dissolved in dimethyl sulfoxideand diluted with PBS (a total volume of 5 ul) to be injected into the right ventricle via a Hamilton syringe (bregma: ML = -1.8 mm, AP = -0.9 mm, and DV = 3.5 mm deep from the dura) [26]. The equal volume of PBS was injected into the right ventricle of rats in the Model group. The rats were examined via forced swimming (FST), tail suspension (TST), and open eld (OFT) tests. In the end, the brains of rats were used for various molecular (n=8 in each group) and histological (n=4 in each group) studies. The fresh samples of the hippocampus were extracted immediately after sacri cing and put in a freezing tube and kept at -80̊ C . Also, for histological studies, the total brain was isolated after pre xation. The pre xation procedure was done via the transcranial perfusion of normal saline to remove the blood from the brain, followed by 4% paraformaldehyde perfusion (PFA, Sigma) via the same route. The post-xed was done by using a 10% formalin at 4° C for 72 h.

Forced swimming test (FST)
A glass cylinder with a height of 80 cm and a diameter of 30 cm lled with 40 cm of water (25°C) was used to evaluate the rats' behavior for two consecutive swimming sessions of training and test. The cylinder was lled with tap water (23 ± 1 °C), and water depth was adjusted according to the animal size.
Therefore, rats could not touch the bottom of the cylinder. At rst, for training, each rat was placed in the cylinder for 10 minutes and forced to swim. After 24 h, the procedure was repeated for 5 min period as a test session. In this session, the animal behavior was video-recorded by a blind observer, and the immobility, latency, swimming, and climbing times were recorded [30]. An enhanced time of immobility time was considered as depressive-like behavior.

Tail Suspension Test (TST)
One day after FST, TST was performed. Brie y, each rat was suspended via its tails using adhesive tape to a horizontal bar for 6 min (2 min for adaptation and 4 min for test). The test was performed by a blind observer, and the immobility time was recorded.
Open Field Test (OFT) The OFT was performed to evaluate the depressive-like behavior, as reported previously [31]. Brie y, the open-eld apparatus (80 cm×80 cm×50 cm 2 ) is consists of oor divided into 25 equal squares. Each rat was placed in the center of the open eld individually and allowed to freely explore for 5 min, and the behavior was recorded. The time of immobility was evaluated for a 5 min period. After nishing the test, the rat was placed in the home cage by the experimenter. After each test, the apparatus was cleaned with 90% ethanol to remove olfactory cues.

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
The fresh samples were used for quantitative real-time PCR (qRT-PCR) assay (n= 4 in each group) of NF-κB, NLRP3, Caspase-1, and ASC. In summary, total RNA was isolated from samples by using the Tripure Isolation Reagent (Roche Applied Science, Peuzberg, Germany) according to the instruction. NanoDrop™ 2000/2000c spectrophotometer (Thermo Fisher Scienti c) was used for evaluation of its purity. For complementary DNA synthesis, PrimeScript RT Reagent Kit (Takara Bio Inc., Otsu, Shiga, Japan) was used for reverse transcription. The PCR primers used are shown in Table 1. The StepOnePlus Real-Time PCR machine was carried out to run the reactions in triplicates. beta-actin (β-actin) was used to normalize the gene expression, and the relative fold change expression of genes was calculated via the 2-ΔΔCt method [32].

Western Blot
The isolated hippocampal samples were used for western blot (n = 4 in each group) to determine the synthesis of in ammasome proteins. The samples were lysed via a lysis buffer (RIPA) and centrifuged. Total Protein Kit, Micro (Sigma, USA) was used to detect the total protein concentration. After protein denaturation, 5 μg of protein was loaded on 10% SDS-PAGE, and separated proteins were put on polyvinylidene di uoride transfer membranes (Sigma, USA) and then incubated for one hour with speci c primary antibodies (Novus Biologicals, USA). Then, the procedure was followed by incubation of Membranes for one hour with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, Germany). Protein bands were detected by the luminescent substrate solution (Sigma, USA). Finally, to quantify the speci c bands, ImageJ software (NIH, USA) was used. GAPDH (Thermoscienti c, USA) was used for normalization [33].

Nissl Staining (Cresyl Violet Staining)
After xation and tissue processing (n = 4 samples in each group), 5-um-thick coronal sections cut by microtome (Leica Biosystems, Milan, Italy) and mounted on the slides. After subjecting to Nissl staining, four random sections were picked for light microscopy observation. The photographs were prepared under an optical microscope (Labomed, USA). The photomicrographs were prepared from the CA1 region, and the number of dark neurons (intensely stained neurons) were calculated in each photomicrograph.

Statistical analysis
Data were analyzed via SPSS software (V22.0) and expressed as mean ± SD. The analyses were carried out using the one-way analysis of variance (ANOVA) and followed by post hoc Tukey's comparison. P value less than 0.05 was considered as signi cant.

Behavioral tests
To determine the effects of T3 on depressive-like behaviors, we performed behavioral investigations,

Western Blot Analysis
The mean protein levels of NF-κB, NLRP3, Caspase-1, and ASC were investigated in the hippocampal region. Fig. 4 shows that there was a signi cant increase in the protein levels of hippocampal NF-κB, NLRP3, ASC, and Caspase-1 in the Model and Model+T3 groups compared to the Control group (P< 0.05, g. 4, a-e). The protein levels of hippocampal NLRP3, Caspase-1, ASC, and signi cantly reduced in the Model+T3 group compared with the Model group (P < 0.05, g. 4, a-e).

Histopathological Examination
For the evaluation of pathological changes, Nissl staining was performed. The pathological alterations of CA1 were investigated in terms of the number of dark neurons and the pyramidal cell layer thickness. A signi cantly increased number of dark neurons in the CA1 region was reported in the Model and Model+T3 groups compared to the Control group (P < 0.05, g. 5, a-d). Also, the number of dark neurons reduced in the Model+T3 group compared to the Model group (P < 0.05, g. 5, a, b, c, & d). The pyramidal cell layer thickness signi cantly decreased in the CA1 region in the Model and Model+T3 groups compared to the Control group (P < 0.05, g. 5, a, b, c, & e). In addition, there was a signi cant increase in the pyramidal cell layer thickness in the Model+T3 group compared to the Model group (P < 0.05, g. 5, a, b, c, & e).

Discussion
The results of the current study revealed that treatment with a single dose of T3 ameliorated the depression-like behaviors induced by restraint stress. The IVC microinjection of T3 could alleviate the activity of in ammasome via reducing the gene expression and protein concentrations of NF-κB, NLRP3, ASC, and Caspase-1 in the hippocampus. Additionally, T3 improved the histopathological changes of the CA1 hippocampal region.
In the present study, we exposed the animals to chronic immobility stress to induce depressive-like behavior. According to the ndings, chronic stress increased the immobility behavior of animals. Also, the enhanced levels of NF-κB, NLRP3, ACs, and Caspase-1 were observed in the hippocampus. Pathologically, the number of dark neurons and the pyramidal layer thickness decreased in the Model group. These ndings indicated that neuroin ammation after animals to the stressor induced the histopathological and behavioral alterations in animals. Rodent models of chronic restraint stress with more than 35 years of use for modeling psychological disease have been employed in the investigation of biological and behavioral manifestations of clinical depressive disorders in humans [34,35]. Depression is a psychological disorder associated with several pathological events, such as neural death in the hippocampus [36]. The neural loss in the hippocampus is an important factor in decreasing the hippocampal volume in depressive disorders [37]. According to the literature, psychological or physical stressors may be followed by the activation of in ammation and subsequent production of in ammatory cytokines, resulting in functional and structural changes in neurons [38]. Several studies have demonstrated that the interaction between the CNS and the immune system plays a critical role in stressinduced neuroin ammation and depression [39]. Therefore, peripheral administration of immunostimulants in animal models is often used for the induction of in ammation-related depression [40]. The anxious and depressive behaviors have been shown to be associated with the enhanced levels of TNF-α in the hippocampus and striatum [41].
Among a wide range of cytokines, IL-1β seems to play an important role in the pathological features of depressive-like behavior caused by stress [42]. It has been demonstrated that IL-1β reduced the neurogenesis in human hippocampal progenitor cells through stimulation of the kynurenine signaling pathway, a common nding in depression. Both inhibitors of this pathway and traditional antidepressants can modulate this effect [43]. The NLRP3 in ammasome is the major intercellular mediator of IL-1β maturation and secretion [44]. The activation the NF-κB pathway and NLRP3 in ammasome has been proven in animal models of depression and patients with major depressive disorders [45,46]. Furthermore, NF-κB signaling and NLRP3 in ammasome have previously been suggested as a link between the immune system and stressors, the potential pathologic features in the development of depressive disorder [14,46]. Activation of NF-κB induces the transcription of pro-IL-18, pro-IL-1β, and NLRP3 [47]. Besides, some factors, e.g., overload calcium, reactive oxygen species, ATP can directly stimulate NLRP3 in ammasome [48,49]. On the other hand, in ammation is commonly related to oxidative stress. It seems that oxidative damage, such as DNA oxidation and lipid peroxidation, contribute to the pathological features of neuropsychiatric diseases [50]. Additionally, the association between glutamate and in ammation has been well demonstrated [35], as a large number of clinical studies suggest that pathophysiology of depression is associated with dysfunction of the predominant glutamatergic system, known as the glutamate hypothesis [51]. The NLRP3 in ammasome also is responsible for pyroptosis and apoptosis in hippocampus neurons mediates depressive-like behavior in diabetic mice [52]. Therefore, the increased number of dark neurons are associated with enhanced activation of NF-κB/ NLRP3 in ammasome pathway.
In the current study, we used a single dose of IVC T3 following the induction of depressive-like behaviors. The administration of T3 could inhibit the activation of the in ammasome complex in the hippocampal region via reducing the NF-κB, NLRP3, ASC, and Caspase-1 concentration and prevent the pathological features seen in the hippocampal region. These alterations were correlated with improved depressive-like behavior according to FST, TST, and OFT ndings, suggesting that T3 could survive the neurons of the CA1 region via anti-in ammatory properties. According to the evidence from preclinical and clinical THs, abnormalities are seen in depressive disorders [53,54]. Therefore, the regulation of the thyroid axis may help to improve the symptoms of depression. T3 alone or in combination with antidepressants has been used for the treatment of patients with depressive disorders [54]. THs are essential for brain development in both fetal and post-natal periods [24]. As previously recorded in mammalian brain researches, the levels of T3 and T3/T4 ratio are higher in the brain compared to the systemic circulation [55]. T3 has been shown to augment or accelerate the treatment of depressive disorders [56]. This hormone acts as a stimulator of gene expression and regulates the cellular energy for metabolism and neurogenesis in the CNS [57]. Recently, IVC injection of T3 was revealed to survive neurons of the CA1 hippocampal region in the rat model of brain stroke via the upregulation of neurotrophic factors [58]. In another study, exogenous T3 exerted neuroprotective features via modulation of the NF-κB pathway (anti-in ammatory effects) and enhanced levels of neurotrophic factors in a traumatic brain injury [59]. In addition, the administration of T3 could diminish alcoholic liver disease and ischemia-reperfusion-induced liver via modulation of the NLRP3 signaling pathway [60, 61]. In accordance with our results, these ndings con rm that T3 of may suppress the NF-κB/ NLRP3 in ammasome pathway via anti-in ammatory features, contribute to reverses the neural death in hippocampus, and improve the depressive like behavior in rats exposure to the chronic stress.

Conclusion
In summary, restraint stress induction activated the NLRP3 in ammasome in the hippocampus region, correlated with pathological and behavioral alterations. To attenuate these changes, we used a single dose of T3 via the IVC route. The ndings of the current study indicated that T3 regulated the gene expression and protein levels of NF-κB, NLRP3, ASC, and Caspase-1 in the hippocampus and improved the pathological and behavioral alterations induced by restraint stress in the rat model. According to the results, T3 could survive the pyramidal neurons of the hippocampus via regulation of e NF-κB/ NLRP3 in ammasome pathway and con rm the bene cial effects of T3 in the treatment of depressive symptoms.