Effect of imipramine on memory, adult neurogenesis, neuroinflammation, and mitochondrial biogenesis in a rat model of alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive decline and memory loss. Imipramine, a tricyclic antidepressant, has potent anti-inflammatory and antioxidant properties in the central nervous system. The aim of this study was to investigate the neuroprotective effects of imipramine on streptozotocin (STZ)-induced memory impairment. Male Wistar rats received an intracerebroventricular injection of STZ (3 mg/kg, 3 μ l/ventricle) using the stereotaxic apparatus. The Morris water maze and passive avoidance tests were used to evaluate cognitive functions. 24 h after the STZ injection, imipramine was administered intraperitoneally at doses of 10 or 20 mg/kg for 14 consecutive days. The mRNA and protein levels of neurotrophic factors (BDNF and GDNF) and pro-inflammatory cytokines (IL-6, IL-1 β , and TNF-α ) were measured in the hippocampus using real-time PCR and ELISA techniques, respectively. In addition, real-time PCR was used to evaluate the mRNA levels of markers associated with neurogenesis (Nestin, DCX, and Ki67) and mitochondrial biogenesis (PGC-1 α , NRF-1, and TFAM). The results showed that imipramine, especially at a dose of 20 mg/kg, effectively improved STZ-induced memory impairment. This improvement was associated with an increase in neurogenesis and neurotrophic factors and a decrease in neuroinflammation and mitochondrial biogenesis dysfunction. Based on these results, imipramine appears to be a promising therapeutic option for improving cognitive functions in neurodegenerative diseases such as AD.


Introduction
Alzheimer's disease (AD), the most common form of dementia, is a multifactorial pathology characterized by amyloid beta (Aβ) accumulation, neuroinflammation, oxidative stress, mitochondrial dysfunction, and cognitive impairment (Chavoshinezhad et al., 2023;Thakral et al., 2023).Impaired adult neurogenesis has also been observed in the brains of AD patients (Babcock et al., 2021) and animal models of AD (Stazi and Wirths, 2021).In the mammalian brain, adult neurogenesis is a multistep process that generates new neurons throughout life.Neurogenesis starts with the proliferation of resident neural stem cells (NSCs) and neural progenitor cells (NPCs) within two neurogenic niches in the central nervous system (CNS): the subventricular zone (SVZ) of the lateral ventricles (LVs) and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus (Denoth-Lippuner and Jessberger, 2021).It has been reported that the newly produced hippocampal neurons are essential for learning, memory, and recovery from neuronal damage (Semënov, 2019).Numerous extrinsic and intrinsic factors have the potential to affect the process of adult neurogenesis.Growth factors and neurotrophins, such as brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF), initiate the process of neurogenesis by promoting the differentiation, maturation, and survival of proliferating NSCs and NPCs.In addition, these factors are critical for regulating axonal growth, synaptic plasticity, and cognitive functions (Numakawa et al., 2018;Ribeiro and Xapelli, 2021).
Neurogenesis has been reported to be inhibited by neuroinflammation, which results from the activation of astrocytes and microglia and the release of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and IL-1β (Kaur et al., 2019).Several studies have shown that the cognitive abnormalities in AD are associated with increased neuroinflammation and decreased adult neurogenesis (Bassani et al., 2018;Kaur et al., 2019).Another major theory about the etiology of AD and other neurodegenerative diseases is mitochondrial dysfunction (Ashleigh et al., 2023).Mitochondrial functions and their dynamic properties, such as mitochondrial biogenesis, are critical for the survival and proper functioning of brain cells (Markaki and Tavernarakis, 2020).Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), which is responsible for mitochondrial biogenesis, activates the expression of several downstream transcription factors, including mitochondrial transcription factor A (TFAM) and nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) (Gureev et al., 2019).Furthermore, it has been shown that disruption of PGC1α activity and its downstream target genes can lead to neurodegenerative diseases such as AD by impairing mitochondrial biogenesis (Panes et al., 2022).
Imipramine is a tricyclic antidepressant that increases the synaptic activity of serotonin and norepinephrine by inhibiting their reuptake (Kandil et al., 2016).In addition to its antidepressant effects, imipramine has been shown to be neuroprotective in several models of neuronal cell death (Lieberknecht et al., 2020;Ono et al., 2012;Peng et al., 2008).Imipramine has also been reported to increase neurogenesis and improve cognitive function by increasing basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and BDNF in the mouse brain (Han et al., 2011;Rantamaki et al., 2007;Sharma et al., 2016).Furthermore, other studies have shown that imipramine reduces Aβ accumulation, tau hyperphosphorylation, damage to synaptic proteins, microglia and astrocytes activation, neuronal death, and cognitive deficits in some in vitro and in vivo models of AD (Crivelli et al., 2023;Javadpour et al., 2021;Johnson et al., 2022;Wuwongse et al., 2013).
Although previous studies have well demonstrated the antiinflammatory and antioxidant effects of imipramine (Chavant et al., 2010;Kandil et al., 2016), however, its protective effects against AD have been less studied.Therefore, the aim of the present study was to investigate the potential neuroprotective effects of imipramine on cognitive functions, neurogenesis, neuroinflammation, and mitochondrial biogenesis in a rat model of AD induced by intracerebroventricular injection of streptozotocin (ICV-STZ), a valid animal model of AD (Akhtar et al., 2020).

Animals
Adult male Wistar rats weighing 230-270 g were used in this study.Rats were randomly housed in groups of four in a standard polypropylene cage at a temperature of 21 ± 2 • C, humidity of 50 ± 10 %, and a 12-h light/dark cycle.They had access to tap water and a standard laboratory chow diet.The NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996) was followed for all experimental procedures.In addition, all experimental procedures were approved by the Ethics Committee of Kharazmi University (IR.KHU.REC.1401.031).

Experimental animal groups and protocol design
Six experimental groups (n = 8 in each group) were randomly selected from the rats as follows: (1) Control group: Rats were injected with ICV-normal saline 0.9 % (NS) (vehicle of STZ; 3 μl/ventricle) on day 0 and treated with intraperitoneal (IP)-NS (vehicle of imipramine) from days 1 to 14.The doses of STZ (Sigma-Aldrich, St. Louis, MO, USA; purity ≥98 %) (Akhtar et al., 2020) and imipramine (Sobhan Darou, Rasht, Iran; purity ≥99.8 %) (Javadpour et al., 2021;Réus et al., 2010) were selected based on previous studies.The Morris water maze (MWM) and passive avoidance (PA) tests were used to assess cognitive abilities.After the behavioral assessments, all rats were killed and hippocampal tissues were immediately removed for the real-time polymerase chain reaction (Real-time PCR) and enzyme-linked immunosorbent assay (ELISA) (n = 4/group).Fig. 1a shows the schematic design of this study.

Microinjection of streptozotocin (STZ)
Rats were deeply anesthetized with a combination of ketamine (80 mg/kg) and xylazine (20 mg/kg) and placed in a stereotaxic apparatus (Stoelting, USA).After exposure of the skull, the stereotaxic coordinates for ICV injection were determined: anteroposterior = − 0.8 mm from the bregma, mediolateral = ±1.5 mm from the midline, and dorsoventral = − 3.6 mm from the skull (Paxinos and Watson, 2007).Before injection, STZ (3 mg/kg) was freshly dissolved in NS and injected into the ventricles using a 10 μl Hamilton syringe at a flow rate of 1 μl/min.To prevent reflux, the needle was left in place for an additional 2 min after delivery.After surgery, the rats were placed in separate cages to recover from anesthesia and then housed in groups of four rats per cage.

Morris water maze (MWM) test
This test was performed to assess spatial learning and memory (Goel et al., 2015).The maze consisted of a circular pool that was black in color and had dimensions of 150 × 60 cm.It was filled with water at a temperature of 24 ± 1 • C, and the water level was maintained at a height of 45 cm.The maze was divided into four equal quadrants, and in the target quadrant, an escape platform (38 cm high and 11 cm in diameter) was placed in the center, 2 cm below the water surface, and maintained there during the experiment.One day before the start of the training trials (day 14), the rats were habituated to swim in the maze without the platform for 60 s.From day 15, the rats were exposed to four training trials per day for a period of 4 days, with an inter-trial interval of 10 min.In each trial, rats were separately placed in one of the quadrants facing the pool wall and given 60 s to locate the platform.Escape latency (time to find the platform) was recorded when the rats successfully found the ventricle) and imipramine (10 or 20 mg/kg, once a day for 14 days, 24 h after STZ) administration on body weight on days 0, 7, and 14.No significant changes were observed within or between groups (p > 0.05).Data are reported as mean ± SEM (n = 8/group).
A.J. Hasanabadi et al. location of the platform within 60 s.After 24 h, the rats were subjected to a 60 s probe trial by removing the platform.During this trial, the latency to the first cross from the former platform position, the number of crossings from the former platform position, and the time spent in the target quadrant were recorded.The rats' movements were recorded by a camera (Panasonic Co., Japan) attached to a computer running Ethovision software (version XT7, The Netherlands) throughout training and testing days.

Passive avoidance (PA) test
This test was used to evaluate memory retention deficits in accordance with the previously described method (Tamijani et al., 2019).The device was a two-compartment shuttle box with a guillotine door separating the dark and light compartments.In summary, rats were placed in the light component 30 min after the habituation trial, and 10 s later the door was opened to allow rats to enter the dark component.The door was then closed and the component's floor-mounted electrified steel bars immediately delivered a foot shock (1 mA, 50 Hz, and 2 s).After 20 s, the door was opened to allow the rat to enter the light component.If the rat did not enter the dark component within 120 s, an inhibitory avoidance response was successfully recorded; otherwise, the rat received another shock.Retention trials were evaluated after 90 min and 24 h and were considered short-term memory (STM) and long-term memory (LTM), respectively.These trials were performed in the same manner as the training trial, except that no foot shock was delivered and the latency into the dark component, the step-through latency, was recorded for 300 s and used as an index of retention of the training experience.

Real-time polymerase chain reaction (real-time PCR)
Total RNA was extracted from hippocampal tissues using the YTzol reagent (Yekta Tajhiz Azma, #YT9063).The purity of RNA was verified spectrophotometrically using Nano-Drop (Thermo Fisher Scientific, USA).The isolated RNA was reverse transcribed into cDNA using the PrimeScriptTM RT Reagent Kit (Takara, Japan, #RR037A) according to the manufacturer's instructions.The cDNA was then used as a template to evaluate the expression levels of the genes doublecortin (DCX), Nestin, Ki67, BDNF, GDNF, TNF-α, IL-1β, IL-6, PGC-1α, NRF-1, and TFAM.SYBR Green real-time PCR Master Mix (2×) (Ampliqon, Denmark) and ABI StepOne equipment (Applied Biosystems, USA) were used for the real-time PCR under the following cycle conditions: 10 min at 95 • C (activation), 15 s at 95 • C (denaturation), 30 s at 60 • C (annealing), and 30 s at 72 • C (extension).β-actin was used as a housekeeping gene to normalize all genes.The relative changes in expression of the above genes were calculated using the 2 -ΔΔCt method (Livak and Schmittgen, 2001).The primer sequences are shown in Table 1.

Statistical analysis
The 16th version of SPSS software was used to perform all statistical analyses.Two-way repeated measures analysis of variance (ANOVA) with Tukey's post hoc test was used to evaluate the results of body weight and MWM training days.The results of the MWM probe session, PA test, and molecular studies were evaluated using one-way ANOVA.The mean ± SEM (standard error of the mean) was used to express all data, and the value of P < 0.05 was considered statistically significant.

Imipramine alleviated deficits in spatial learning and memory, as well as short-and long-term memory, in the STZ-injected rats
Two-way repeated measures ANOVA showed that the effects of treatment [F (5, 42) = 15.72,P < 0.001] and time [F (2.286, 123.5) = 132.817,P < 0.001] on the escape latency were statistically significant during the training days of MWM.In addition, between-group analysis revealed that the STZ group had a longer escape latency than the control group on training days 1-4 (Fig. 2a, P < 0.05, P < 0.001, P < 0.001, and P < 0.01, respectively), indicating a decrease in learning performance.However, administration of imipramine (10 or 20 mg/kg) to STZinjected rats resulted in a significant decrease in the escape latency on days 2-4 compared to the STZ group.The rats that received imipramine alone (10 or 20 mg/kg) did not exhibit a considerable difference in the escape latency compared to the control group (Fig. 2a, P > 0.05).In addition, the swimming speed of the experimental groups during the training days was not statistically significant (data not shown).In the probe session, one-way ANOVA indicated that the STZ group exhibited a decrease in the time spent in the target quadrant [F (5, 42) = 6.459,P <
Moreover, as shown in Fig. 2e and f, one-way ANOVA indicated that the STZ-injected rats exhibited a decreased latency entrance into the dark component compared to the control group in the STM [F (5, 42) = 5.006, P < 0.001] and LTM [F (5, 42) = 4.053, P < 0.01] sessions, indicating a decline in both short-and long-term memory, respectively.In both sessions, STZ-injected rats treated with imipramine (20 mg/kg) showed a significant increase in the step-through latency compared to the STZ group (P < 0.01 and P < 0.05, respectively).During these sessions, the rats that received imipramine alone (10 or 20 mg/kg) did not display a remarkable difference in the step-through latency compared to the control group (P > 0.05).
Taken together, the MWM and PA results showed that imipramine treatment reduced STZ-induced disorders in spatial learning and memory as well as short-and long-term memory at a dose of 20 mg/kg as an effective dose.Thus, this dose was selected for the molecular assays based on the behavioral data.

Imipramine treatment reduced neuroinflammation in the hippocampus of STZ-injected rats
The mRNA and protein levels of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) in rat hippocampus were evaluated by real-time PCR and ELISA.One-way ANOVA revealed that STZ significantly increased the mRNA and protein levels of IL-6 [F (3, 12) = 12.510, P < 0.001 and F Fig. 3.The effects of imipramine treatment (20 mg/kg, once a day for 14 days, 24 h after STZ) on the impairment of neurogenesis and neurotrophic factors induced by STZ (3 mg/kg, 3 μl/ventricle).Real-time PCR analysis of (a) Nestin, (b) DCX, and (c) Ki67, as well as real-time PCR and ELISA analyses of (d-e) BDNF and (f-g) GDNF in the hippocampus of rats.Data are reported as mean ± SEM (n = 4/group).One-way ANOVA followed by Tukey's post hoc test was used for statistical analysis.*P < 0.05, **P < 0.01, and ***P < 0.001 vs. control group; # P < 0.05 and ## P < 0.01 vs. STZ group.

Discussion
Our findings showed that ICV-STZ resulted in cognitive deficits, reduced neurogenesis and neurotrophic factors, increased levels of proinflammatory cytokine, and impaired mitochondrial biogenesis in the hippocampus of rats.Whereas imipramine treatment, especially at a dose of 20 mg/kg, effectively reduced these STZ-induced adverse effects in both behavioral and molecular assessments.

Cognitive functions
ICV-STZ is widely used in experiments to create animal models of AD (Akhtar et al., 2020).Several studies have indicated that ICV-STZ, particularly at a sub-diabetogenic dose (3 mg/kg), given once (on day 1) or twice (on days 1 and 3), leads to cognitive impairments two weeks after injection, and these disorders can persist for 3 or 4 weeks after injection (Akhtar et al., 2020;Isik et al., 2016;Mehla et al., 2013).In this study, we found that ICV-STZ (3 mg/kg) significantly impaired spatial learning and memory as well as short-and long-term memory two weeks after injection.Consistent with our findings, numerous studies have shown that ICV-STZ-induced animal models of AD exhibit learning and memory impairments, as well as histological and biochemical changes in the brain areas associated with cognitive functions (Akhtar et al., 2020;Gerzson et al., 2020;Isik et al., 2009;Isik et al., 2016;Sharma and Garabadu, 2020).
In this study, we also found that treatment with imipramine (once daily for 14 consecutive days, 24 h after STZ) significantly ameliorated cognitive deficits in the STZ-injected rats, especially at a dose of 20 mg/ kg.In agreement with our findings, Chavant et al. (2010) have indicated that chronic imipramine administration decreases cognitive impairment by inhibiting TNF-α expression and Aβ accumulation in the hippocampus and frontal cortex of a mice model of AD induced by ICV injection of Aβ25-35. Moreover, Javadpour et al. (2021) have shown that imipramine alleviates spatial learning and memory deficits in the STZ-induced sporadic AD rat model by increasing IRS-1 and ERK activity and decreasing caspase-3, P38, and JNK activity in the hippocampus.In addition, Johnson et al. (2022) have shown that imipramine reduces apolipoprotein E4-induced Aβ pathology in primary neurons from 5xFAD transgenic mice and inhibits phosphorylated tau neuropathology in primary neurons from TgF344-AD transgenic rats.They also showed that these effects can be associated with improvement in cognitive functions.Furthermore, Erburu et al. (2015) have indicated that imipramine improves cognitive performance in a mouse model of chronic mild stress.Lee et al. (2022) have also shown that imipramine reduces Fig. 4. The effects of imipramine treatment (20 mg/kg, once a day for 14 days, 24 h after STZ) on neuroinflammation induced by STZ (3 mg/kg, 3 μl/ventricle).Realtime PCR and ELISA analyses of (a-b) IL-6, (c-d) IL-1β, and (e-f) TNF-α in the hippocampus of rats.Data are reported as mean ± SEM (n = 4/group).One-way ANOVA followed by Tukey's post hoc test was used for statistical analysis.**P < 0.01 and ***P < 0.001 vs. control group; # P < 0.05 and ## P < 0.01 vs. STZ group.traumatic brain injury-induced hippocampal neuronal death, oxidative damage, dendritic loss, glial activation, and cognitive dysfunction in rats.

Neurogenesis
The relationship between learning, memory and adult neurogenesis has been studied extensively in recent years.Several studies have shown that increased hippocampal neurogenesis promotes hippocampusdependent cognitive processes such as spatial learning and memory (Lieberwirth et al., 2016;Yau et al., 2015).In this study, we found a considerable reduction in the mRNA levels of neurogenesis markers such as Nestin, DCX, and Ki67 in the hippocampus of STZ-injected rats.Consistent with our findings, Bassani et al. (2018) have shown that reduced adult hippocampal neurogenesis is associated with impairments in STM and LTM in a rat model of AD induced by STZ.In addition, Qu et al. (2012) have shown a decrease in the number of new neurons in the DG after ICV-STZ.Sun et al. (2015) have indicated that ICV-STZ decreases the production of both immature and mature neurons in the hippocampus of rats three months after injection, and this decrease is associated with the onset of Aβ pathology.Another study has shown that ICV-STZ decreases neural stem cell proliferation, migration, and neuronal differentiation in the SVZ and DG of rats 11 and 18 days after injection and impairs spatial learning and memory in these animals (Mishra et al., 2018).
Several previous studies have reported that antidepressants promote neurogenesis, neuronal regeneration, and synaptic plasticity in the hippocampus, which may affect cognitive functions (Han et al., 2011;Kitamura et al., 2011).In this study, we found that imipramine increased the expression levels of neurogenesis indicators in the hippocampus of STZ-injected rats.Consistent with our results, it has been reported that long-term administration of imipramine improves cognitive functions by promoting hippocampal cell proliferation and neurogenesis after traumatic brain injury in mice (Han et al., 2011).Another study has shown that imipramine increases neurogenesis in the DG of the hippocampus after fluid percussion brain damage in rats (Zhang et al., 2013).Furthermore, it has been indicated that chronic imipramine administration increases neurogenesis and synaptogenesis in the hippocampus of a genetic rat model of depression (Chen et al., 2010).

Neurotrophic factors
Neurotrophic factors have been reported to improve cognitive deficits, reverse neuronal loss, promote neuronal plasticity and neurogenesis, and significantly protect critical brain circuits associated with AD (Nigam et al., 2017).Studies have demonstrated that the expression levels of neurotrophic factors in the hippocampus, frontal and temporal cortices are significantly lower in AD patients and animal models of AD.These studies have also shown a positive correlation between the severity of cognitive deficits and decreased levels of neurotrophic factors (Li et al., 2009;Ng et al., 2019;Numakawa et al., 2018;Ribeiro and Xapelli, 2021).In this study, we found a considerable decrease in the mRNA and protein levels of neurotrophic factors, including BDNF and GDNF, in the hippocampus of STZ-injected rats.In agreement with our findings, Rajasekar et al. (2017) have shown a decrease in the BDNF level in the hippocampus of STZ-induced AD rat model, along with deficits in long-term potentiation and spatial learning and memory.Moreover, Qi et al. (2021) have reported that bilateral hippocampal injection of STZ induces AD-like behaviors in mice and remarkably decreases the protein level of BDNF in the prefrontal cortex and hippocampus of these animals.
Antidepressants, particularly tricyclic antidepressants, have been reported to modulate key signaling pathways involved in cell survival, synaptic plasticity, and cognitive function by increasing the expression of neurotrophic factors (Mosiołek et al., 2021;Poon et al., 2021;Sharma et al., 2016).In this study, our results demonstrated that imipramine increased the mRNA and protein levels of BDNF and GDNF in the hippocampus of STZ-injected rats.Consistent with our results, studies have shown that long-term imipramine administration increases the mRNA level of BDNF and synaptophysin in the hippocampus of rats (Larsen et al., 2008).Wuwongse et al. (2013) have also demonstrated that imipramine reduces damage to synaptic proteins, such as synaptophysin and synaptotagmin, in primary cultures of hippocampal neurons treated with oligomeric Aβ (a model agent for AD).Furthermore, Kandil et al. (2016) have demonstrated that imipramine increases BDNF level in the nigrostriatal system and delays the progression of dopaminergic neuron loss in this system in a rat model of Parkinson's disease.Besides, another study has shown that imipramine can inhibit neuroinflammation and apoptosis induced by lipopolysaccharide and promote serotoninergic differentiation in hippocampal-derived NSCs by activating BDNF and MAPK pathways (Peng et al., 2008).Furthermore, long-term administration of imipramine has been shown to reverse chronic stress-induced down-regulation of BDNF in both preclinical and clinical studies (Poon et al., 2021).All these results indicate that neurogenesis and neurotrophic factors play an important role in cognitive functions; therefore, it is possible that imipramine may reduce cognitive impairments in the STZ-injected rats by enhancing neurogenesis and neurotrophic factors.

Neuroinflammation
Activation of glial cells triggers the release of pro-inflammatory cytokines, which initiate inflammatory responses and contribute to neuronal cell death in AD (Bassani et al., 2018;Kaur et al., 2019).In the present study, we observed an increase in the mRNA and protein levels of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, in the hippocampus of STZ-injected rats, indicating a neuroinflammatory response.In agreement with our results, many studies have shown an increase in reactive gliosis and pro-inflammatory cytokines in the brain of STZ-induced animal models of AD, which are associated with increased neuroinflammation, reduced adult neurogenesis, and cognitive impairment (Bassani et al., 2017;Bassani et al., 2018;Mishra et al., 2018;Qi et al., 2021).
Furthermore, our study showed that imipramine effectively reduced the mRNA and protein levels of pro-inflammatory cytokines in the hippocampus of STZ-injected rats.In agreement with our findings, it has been demonstrated that imipramine considerably prevents cognitive decline and reduces TNF-α levels in the brain of a mice model of AD induced by Aβ25-35 (Chavant et al., 2010).Crivelli et al. (2023) have also found that administration of imipramine to 5xFAD mice reduces Aβ accumulation, neuronal death, and neuroinflammationin the brain of these animals.Another study has also shown that long-term imipramine treatment reduces oxidative damage, microglial activation, and expression of pro-inflammatory cytokines in a rat model of PD (Kandil et al., 2016).Other studies have indicated that imipramine reverses anxiety and depression-like behaviors as well as ameliorates the neuroinflammatory responses in the peripheral and central nervous systems of mice (Ramirez et al., 2015;Ramirez and Sheridan, 2016).Given that neuroinflammation has an adverse effect on adult neurogenesis and that both neuroinflammation and impaired neurogenesis lead to cognitive deficits, it is reasonable that imipramine may ameliorate cognitive deficits in the current study by reducing inflammation and increasing neurogenesis in the STZ-injected rats.

Mitochondrial biogenesis
Abnormalities in mitochondrial functions, such as mitochondrial biogenesis, have been reported in both AD patients (Wang et al., 2020) and animal models of AD (Singulani et al., 2020).In this study, we observed that rats exposed to STZ showed a decrease in the mRNA levels of PGC-1α, NRF-1, and TFAM, the key regulators of mitochondrial biogenesis.Consistent with these findings, some studies have demonstrated that ICV-STZ induces mitochondrial dysfunction in several brain areas of the experimental animals, which contributes significantly to cognitive impairment (Akhtar et al., 2021;Paidi et al., 2015).Moreover, Singulani et al. (2020) have demonstrated that the levels of NRF-1, TFAM, and PGC-1α are significantly reduced in the hippocampal CA1 and DG of 3xTg-AD mice.In addition, Gong et al. (2013) have found that a decrease in PGC-1α expression may play an important role in the development of abnormal Aβ production, which may lead to the cognitive impairment and neurodegeneration observed in AD patients.Taken together, the enhancement or induction of mitochondrial biogenesis may be considered a novel therapeutic target for many neurodegenerative diseases such as AD (Uittenbogaard and Chiaramello, 2014).
There is a scarcity of research on the effect of antidepressants on mitochondrial biogenesis.Interestingly, in this study, we observed that imipramine increased the mRNA levels of factors involved in mitochondrial biogenesis in the hippocampus of STZ-injected rats.Filipovic et al. (2017) have shown that treatment with fluoxetine, an antidepressant belonging to the class of selective serotonergic receptor blockers, increases mitochondrial activity in several metabolic pathways in the hippocampus of rats.Another study has shown that long-term fluoxetine treatment normalizes depression-like disorders caused by prenatal stress procedures and increases the expression of proteins associated with mitochondrial biogenesis as well as learning and memory in the hippocampus of adult rats (Głombik et al., 2017).Since a number of studies have shown a connection between mitochondrial dysfunction and cognitive disorders (Ashleigh et al., 2023), it is plausible that imipramine ameliorates cognitive deficits in the current study by increasing the expression of factors involved in mitochondrial biogenesis.
Although several histological studies have shown that ICV-STZ induces Aβ accumulation, tau hyperphosphorylation, neuronal loss, neuroinflammation, and mitochondrial dysfunction (as AD biomarkers) in various brain regions (Alluri et al., 2023;Fan et al., 2022;Isik et al., 2009;Isik et al., 2016;Paidi et al., 2015;Wu et al., 2018), however, the effects of imipramine on AD biomarkers in animal models of AD have been less studied (Chavant et al., 2010;Crivelli et al., 2023;Johnson et al., 2022;Wuwongse et al., 2013).Therefore, further studies are needed to evaluate the efficacy of imipramine on the most important biomarkers of AD, especially at the histological levels, in different brain regions of STZ-injected rats.

Conclusions
The present study demonstrated that long-term administration of imipramine, especially at a dose of 20 mg/kg, effectively ameliorated STZ-induced cognitive deficits, at least in part, by enhancing neurogenesis and neurotrophic factors and reducing neuroinflammation and mitochondrial biogenesis impairment.Thus, imipramine is proposed as a potential therapeutic candidate for neurodegenerative diseases such as AD.

Fig. 2 .
Fig. 2. The effects of imipramine treatment (10 or 20 mg/kg, once a day for 14 days, 24 h after STZ) on spatial learning and memory as well as short-and long-term memory impairments induced by STZ (3 mg/kg, 3 μl/ventricle).(a) Escape latency during the training days, (b) time spent in the target quadrant, (c) the number of crossings from the previous platform location, (d) the latency to the first crossing from the previous platform location, (e) the first retention trial 90 min after the acquisition trial (STM), and (f) the second retention trial 24 h after the acquisition trial (LTM).Data are reported as mean ± SEM (n = 8/group).Differences between groups were determined by ANOVA followed by Tukey's test.*P < 0.05, **P < 0.01, and ***P < 0.001 vs. control group; # P < 0.05 and ## P < 0.01 vs. STZ group.