Noopept; a nootropic dipeptide, modulates persistent inflammation by effecting spinal microglia dependent Brain Derived Neurotropic Factor (BDNF) and pro-BDNF expression throughout apoptotic process.

There are largely unknown associations between changes in pain behavior responses during persistent peripheral inflammation and spinal cell alteration such as apoptosis. Some evidence suggests that microglia and microglia related mediators play notable roles in induction and maintenance of central nervous system pathologies and inflammatory pain. By considering those relationships and microglia related nootrophic factors, such as the Brain Derived Neurotrophic Factor (BDNF) in CNS, we attempted to assess the relationship between microglia dependent BDNF and its precursor with pain behavior through spinal cell apoptosis as well as the effect of Noopept on this relationship. Persistent peripheral inflammation was induced by a single subcutaneous injection of Complete Freund's Adjuvant (CFA) on day 0. Thermal hyperalgesia, paw edema, microglial activity, microglia dependent BDNF, pro-BDNF expression, and apoptosis were assessed in different experimental groups by confirmed behavioral and molecular methods on days 0, 7, and 21 of the study. Our findings revealed hyperalgesia and spinal cell apoptosis significantly increased during the acute phase of CFA-induced inflammation but was then followed by a decrement in the chronic phase of the study. Aligned with these variations in spinal microglial activity, microglia dependent BDNF significantly increased during the acute phase of CFA-induced inflammation. Our results also indicated that daily administration of Noopept (during 21 days of the study) not only caused a significant decrease in hyperalgesia and microglia dependent BDNF expression but also changed the apoptosis process in relation to microglia activity alteration. It appears that the administration of Noopept can decrease spinal cell apoptosis and hyperalgesia during CFA-induced inflammation due to its direct effects on microglial activity and microglia dependent BDNF and pro-BDNF expression.

Introduction

In the case of persistent pain states, mounting evidence has illustrated that the release of a variety of inflammatory mediators, such as cytokines and growth factors from neuronal and non-neuronal cells play a crucial role in the induction of inflammatory pain [1]. Although numerous efforts have been made to understand the cellular and molecular basis of chronic pain, its physiological aspects remain mostly unknown [2, 3]. Multiple mechanisms contribute to inflammatory pain, each of which is subject to cellular and molecular alterations of nociceptive neurons in the dorsal horn of the spinal cord. Some mechanisms also underwrite peripheral inflammation, which is accompanied by inflammatory symptoms such as hyperalgesia and edema. Cell apoptosis, as an important consequence of central nervous system (CNS) pathological conditions, is induced by the increment of inflammatory mediators such as cytokines and growth factors [4, 5]. Data from diverse animal models support the idea that apoptosis can induce neuronal sensitization and hyperalgesia [6, 7]. It is noteworthy that these irreversible processes might be accompanied by different nervous system damages such as neuropathic pain [8]. In this regard, Kim et al. showed that induction of spinal nerve ligation (SNL) in rats resulted in DRG neuronal apoptosis, which precedes pain behaviors such as mechanical allodynia [9]. Microglia, as resident macrophages within the CNS, are involved in mediating inflammatory processes and can release pro-inflammatory mediators such as TNF-α [10]. Like other cells like astrocytes, microglia can produce not only pro/anti-inflammatory mediators and neurotrophic factors such as Brain Derived Neurotrophic Factor (BDNF), but can also play neurotoxic or neuroprotective roles in CNS depending on the released mediators [11, 12]. It has become increasingly clear that microglial cells can powerfully control and modulate pain when they are activated in response to peripheral inflammation [13].

Several studies indicate that BDNF is a neurotrophin with important biologic roles in neuronal survival and differentiation [14]. Also, it has a crucial role in regulating inflammatory pain thresholds and secondary hyperalgesia [15, 16]. Previous studies have shown that BDNF synthesis is greatly increased in different populations of DRG neurons in response to peripheral inflammation, and it also has apoptotic effects on neural cells [17, 18]. Groth and Aanonsen showed that BDNF induces an acute dose-dependent thermal hyperalgesia response and also affects central sensitization in NMDA receptor activation-dependent processes [19]. On the other hand, Siuciak et al. presented that midbrain infusion of BDNF can decrease behavioral paw flinch response to subcutaneous formalin injection in both early and late phases of the test [20].

Accumulating evidence supports that Pro-Brain Neurotrophic Factor (pro-BDNF), as a BDNF precursor binds to the p75 neurotrophin receptor (p75NTR) and exerts the opposing biological functions of mature BDNF. Pro-BDNF plays an active role in the regulation of axon pruning, dendritic complexity, and also neuronal apoptosis in the hippocampus. An important piece of the puzzle is missing from the previous studies of BDNF, the microglial activity-dependent pro-BDNF and BDNF relationship with spinal cell apoptosis and pain behaviors during peripheral persistent inflammation [21, 22].

The term nootropics (cognition enhancer) is applied to a group of psychoactive substances that stimulate neuronal functions and enhance cognitive performances such as memory [23, 24]. N-Phenylacetyl-l-prolylglycine ethyl ester (Noopept), a dipeptide analog of piracetam, is one of these synthesized cognition enhancers [25, 26]. There is evidence to suggest that the administration of Noopept can significantly suppress peripheral inflammatory responses to carrageenan [27]. Moreover, it has been shown that Noopept treatment can prevent accelerating neuronal death, which may be related to its capacity to block the neurotoxic effects of calcium ions and glutamate and specifically its anti-inflammatory effects. As such, it deserves consideration as a therapeutic target [28, 29].

In light of the principal role of microglia related mediators in inflammatory pain and cell death induction, the identification of neuroprotective and anti-inflammatory roles of Noopept should generate novel therapeutics for the treatment of inflammatory pain. In the context of inflammatory pain, it is also interesting to consider the relationship between spinal cell apoptosis and the pain process. We, therefore, aimed to investigate the relationships between microglia dependent BDNF and pro-BDNF and spinal cell apoptosis and pain behavioral responses during different stages of CFA-induced inflammatory pain. Moreover, in a second step, we focused on the effects of Noopept administration through this pathway.

Methods

Laboratory animals and experimental procedures

All experimental procedures were performed on adult male Wistar rats (200–220 g). The animals were kept under standard temperature, humidity and lighting condition (22±2 °C, humidity 60–70%, 12h light/dark cycle) with free access to food and water. All procedures were approved by the Ethics Committee of Shahid Beheshti University of Medical Science (IR.SBMU.MSP.REC.1396.818), and were also in accordance with NIH Guidelines for the Care and Use of Laboratory Animals [30]. The rats were divided into five experimental groups as follows: the (a) CFA group (as an inflammatory pain model), (b) CFA + saline (vehicle) group, (c) Mineral oil (CFA control) group, (d) CFA + Noopept (possible treatment option) group, and (e) CFA + Minocycline (non-specific microglia inhibitor) group. According to the study procedure, each group was further divided into 3 subgroups based on different time points in the study (0, 7, and 21 days), and each subgroup included 6 rats. For example CFA group were divided to 3 subgroups: day 0 (CFA0), 7 (CFA7) and 21 (CFA21) and similar classification carried out for CFA + saline, CFA + Minocycline and CFA + Noopept groups (Figure 1A). According to our previous studies, a persistent inflammatory pain model was induced by CFA on day zero (under light anesthesia) in all experimental groups. The mineral oil group received a single subcutaneous injection of sterile mineral oil (100 μL) without the CFA on day zero. Based on our previous studies [31, 32, 33] and others [34, 35], the first week after CFA injection (7 days) is considered as an inflammatory phase, and the third week after CFA injection is considered as an arthritic phase of this model. All treatments began one day after CFA injection. Noopept and Minocycline were administered by daily intraperitoneal (i.p.) injection at 5 mg/kg and 40 mg/kg respectively in different subgroups. Also, saline as a drug solvent was administered by daily i.p. injection. At the end of each period (day 0,7 and 21 days), after conducting behavioral tests (n = 6 per each subgroup), the same rats (n = 3 per each subgroup) were deeply anesthetized with methoxyflurane, decapitated and then the lumbar spinal cord was quickly removed for the detection of expression of spinal proteins. Other rats (n = 6–3 per each subgroup) were anesthetized by i.p. injection of mixed ketamine (100 mg/kg) and xylazine (10 mg/kg). After cardiovascular perfusion using a solution of 4% paraformaldehyde in 0.1 M PBS (pH = 7.4), lumbar spinal cord segments were removed and post-fixed for 48 h at 4 °C in formalin for TUNEL staining (Figure 1B).

Figure 1

A. Study Groups B. Experimental procedures diagram.

Inflammatory pain induction

CFA-induced inflammatory pain was caused by a single subcutaneous injection of (100μL) heat-killed Mycobacterium tuberculosis suspended in sterile mineral oil (10 mg/ml; CFA; Sigma, St Louis, MO, USA) into the rats’ hind paw on day zero under light anesthesia with methoxyisoflurane. The first week after CFA injection was considered the inflammatory phase and the third week was the arthritic phase [36, 37].

Drug administration

In this study, Minocycline (Sigma-Aldrich, Germany), as a microglia activation inhibitor, was diluted in physiological saline (0.9% NaCl) and injected intraperitoneally (i.p.) daily in a dose of 40 ml/kg from the first day after CFA injection at determined time of day (9:00 a.m.) up to day 21 of the study in the CFA + Minocycline group [32]. Noopept (a nootropic dipeptide) was dissolved in saline solution and injected (i.p.) daily in a dose of 5 mg/kg from the first day after CFA injection at determined time of day (9:00 a.m.) up to day 21 of the study in the CFA + Noopept group. Regarding previous studies, we selected the optimal dose of Noopept in our study and it re-suspended 1.25 mg in 500 μl of vehicle to produce a stock solution of 50 mM; that stock was administered daily via i.p. injection [1, 2, 38]. CFA + saline group received saline (as a vehicle of Minocycline and Noopept) in same volume of the other mentioned groups.

Assessment of paw edema

Paw edema assessment was done by measuring paw volume variations during the different stages of the study using a Plethysmometer (model 7141; Ugo Basile, Comerio-Varese, Italy). Briefly, the rats’ hind paws were submerged to the tibiotarsal joint in the electrolyte-filled perspex cell of the plethysmometer. The amount of liquid displaced, which is related to the paw volume, was shown on a digital display. Volume measurements were done twice for each paw, and the average was calculated. Edema was considered by measuring the differences between day zero and the other time points, days 7 and 21 of the study [39].

Thermal hyperalgesia assessment

Paw withdrawal latency (PWL) was used to measure thermal hyperalgesia by applying the Hargreaves test [40]. For this purpose, the rats were placed in Plexiglass chambers (Ugo Basile, Verse, Italy) and habituated for 15 min to the test environment. The heat source was adjusted under the plantar surface of the injected and non-injected hind paws and an infrared light was projected focally, and then PWL was measured. Each paw was tested three times at 5 min intervals and the average value of the withdrawal latency of three consecutive tests was calculated and deducted from the other paw. In the present study, the cut-off time in the absence of a response was 20s.

Western blot

After the behavioral tests, western blot was used to examine the amounts of BDNF, pro-BDNF, Iba1, and Caspase-3 expression in the lumbar spinal cord. Caspase-3 is a commonly activated death protease via the intrinsic and extrinsic apoptotic pathways. This protease is essential for destruction of cellular structures and some typical hallmarks of apoptosis such as chromatin condensation [41]. In this regard, the lumbar part of the spinal cord (L1-L5) was removed and homogenized in a lysis buffer. Protein samples were separated on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12% SDS-PAGE) and transferred to an activated Polyvinylidene difluoride (PVDF) membrane (Roche Diagnostics, Indianapolis, lN, USA), then blocked with 5% Bovine serum albumin (BSA) and incubated overnight at 4 °C with primary antibodies against BDNF (1:1000, ab205067), pro-BDNF (1:1000, NB100-98754), Iba1 (1:1000, ab178846), and Caspase-3 (1; 3000, ab184787). The membrane then was washed three times and incubated for 1h at room temperature with a secondary antibody (1:20000, cell signaling #7074). Finally, the blots were visualized with a chemiluminescence detection system (ECL, Amersham, RPN2235). Band intensity was measured densitometrically using Image J and expressed as the ratio of the intensity of the BDNF, pro-BDNF, Iba1, and Caspase-3 band to β-actin [41].

TUNEL assay

The apoptotic cells in the lumbar section of the spinal cord were detected by TUNEL staining. Briefly, lumbar segments of the spinal cord were embedded in paraffin after fixing in 4% paraformaldehyde for 24h. Then, five slides from each block were prepared for assessing the apoptotic index by means of the TUNEL technique. After deparaffinization in xylene, the sections were rehydrated with different dilutions of alcohol and treated with H2O2 and methanol at room temperature. Afterwards, they were incubated in proteinase K. The TUNEL technique was carried out using a TUNEL-based assay kit (SIGMA, Roche, Germany). The sections were incubated first in horseradish peroxidase (POD) and then in diaminobenzidine (DAB) solution and subsequently counterstained with hematoxylin. Five watch fields were chosen randomly on each section under a microscope (Nikon-E600). Positive brown cells and total cells were counted and analyzed using GraphPad Prism 6 software [42, 43].

Statistical analysis

Data were shown as mean ± standard error of mean (SEM). The statistical analysis used for the behavioral (n = 6 per each subgroup), cellular (n = 3 per each subgroup), and molecular (n = 3 per each subgroup) tests was a one way ANOVA. Statistical comparisons were analyzed by repeated measurements followed by Post hoc Tukey's for evaluation of variants within the groups. An unpaired Student's t-test was used to compare the changes of variants on the same days between the two groups. Statistical significance was accepted at the p ≤ 0.05 level. Statistical analyses were performed using the GraphPad Prism 6.0 program.

Results

Paw volume variation during different stages of CFA-induced inflammatory pain

CFA injection into the rat's hind paw caused a significant increase in paw volume, which persisted until the 21st day of the study. Paw volume showed a significant increase on the 7th and 21st days after CFA injection compared with day zero in the CFA group (p < 0.001). Our data also indicated that paw volume significantly increased on day 21 compared with day 7 after CFA injection in the CFA group (p < 0.001). On the other hand, three weeks administration of Noopept in the CFA + Noopept group significantly reduced paw volume on the 7th (p < 0.001) and 21st (p < 0.001) days of the study compared to the same days in the CFA group. Furthermore, our results revealed that paw volume significantly decreased in the CFA + Noopept group in comparison with the CFA + Minocycline group on days 7 and 21 of the study (p < 0.001). Saline administration, as a vehicle, caused no significant differences in paw volume in comparison with the CFA group at all-time points of the study (Figure 2).

Figure 2

Paw volume variations during different stages of the study. Data is presented as mean ± SEM (n = 6/subgroup). ∗∗∗P < 0.001 for comparing days 7 and 21 of the study with day zero in CFA group. †††P < 0.001 for comparing between days 7 and 21 in the CFA group. ###P < 0.001 for comparing between CFA and CFA + Noopept groups. $$$P < 0.001 for comparing between CFA + Noopept and CFA + Minocycline groups.

Thermal hyperalgesia variations during different stages of CFA-induced inflammatory pain

Our results revealed that hyperalgesia noticeably increased on the 7th day after CFA injection compared with day zero (p < 0.001). However, it significantly decreased on day 21 in comparison with day 7 (p < 0.01) in the CFA group, although hyperalgesia on day 21 was still more than day zero (p < 0.001). Long-term Noopept administration in the CFA + Noopept group significantly reduced thermal hyperalgesia compared with the CFA group on days 7 and 21 (p < 0.001). Moreover, the results illustrated that thermal hyperalgesia significantly decreased in the CFA + Noopept group in comparison with CFA + Minocycline group on days 7 and 21 (p < 0.001). The CFA + Saline (vehicle group) group indicated no significant differences in paw withdrawal latency in comparison to the CFA group at all times of the study (Figure 3).

Figure 3

Thermal hyperalgesia varied in the injected paw during different stages of inflammation. Data is presented as mean ± SEM (n = 6/subgroup). ∗∗∗P < 0.001 for comparing days 7 and 21 of the study with day zero in CFA group. ††P < 0.01 for comparing between days 7 and 21 in the CFA group. ###P < 0.001 for comparing between CFA and CFA + Noopept groups. $$$P < 0.001 for comparing between CFA + Noopept and CFA + Minocycline groups.

Western blot analysis of spinal BDNF expression during different stages of CFA-induced inflammatory pain

Following intraplantar injection of CFA, lumbar spinal BDNF expression demonstrated various changes during the different stages of the study. The results revealed that CFA injection caused significant increase in BDNF expression on day 7 in comparison with day zero (p < 0.001). There was a significant decrease in BDNF expression on day 21 in comparison with day 7 in the CFA group (p < 0.001). Our findings indicated that the administration of Noopept in the CFA + Noopept group caused a significant decrease in spinal BDNF expression on the 7th (p < 0.001) and 21st (p < 0.001) days of study in comparison with the same day in the CFA group. In addition, in comparison with the CFA group, the decrease in BDNF expression in the CFA + Noopept group was significantly more than what was observed in the CFA + Minocycline group on the 7th (p < 0.001) and 21st (p < 0.001) days of study. The CFA + Saline (vehicle) group indicated no significant differences in spinal BDNF expression in comparison to the CFA group at all-time points of the study (Figure 4).

Figure 4

A: Immunoblots of spinal BDNF expression in different experimental groups. (See also Supplementary Figures 1,5). All densitometry data were demonstrated as BDNF/ß-actin ratio. B: Noopept treatment could significantly decrease spinal BDNF expression on day 7th of the study. Data is presented as mean ± SEM (n = 3/subgroup). †††P < 0.001 for comparing between days 7 and 21 in the CFA group. ###P < 0.001 for comparing between CFA and CFA + Noopept groups. $$$P < 0.001 for comparing between CFA + Noopept and CFA + Minocycline groups.

Assessment of spinal pro-BDNF expression during different stages of CFA-induced inflammatory pain

Western blotting analysis of pro-BDNF expression in the lumbar part of the spinal cord showed a significant increase on day 7 in comparison with day zero in the CFA group (p < 0.001). On the other hand, spinal pro-BDNF expression in the CFA group considerably decreased on day 21 compared with days zero (p < 0.05) and 7 (p < 0.001). Our findings also indicated that the Noopept treatment in the CFA + Noopept group significantly decreased pro-BDNF expression on day 7 (p < 0.001 compared with the same day in the CFA group. Based on our results, administration of Noopept in the CFA injected rats was significantly more effective in decreasing pro-BDNF expression compared with the CFA + Minocycline and CFA groups during acute phase of the study (p < 0.001). The CFA + Saline group presented no significant differences in spinal pro-BDNF expression in comparison with the CFA group at all time points of the study (Figure 5).

Figure 5

A: Immunoblots of spinal pro-BDNF expression in different experimental groups (See also Supplementary Figures 2, 5). All densitometry data were demonstrated as pro-BDNF/ß-actin ratio. B: Noopept administration caused significant decrease in spinal pro-BDNF expression in acute phase of the study. Data is presented as mean ± SEM (n = 3/subgroup). ∗P < 0.05 and ∗∗∗P < 0.001 for comparing between 21st and 7th days of the study with day 0 in the CFA group. †††P < 0.001 for comparing between days 7 and 21 in the CFA group. ###P < 0.001 for comparing between CFA and CFA + Noopept groups. $$$P < 0.001 for comparing between CFA + Noopept and CFA + Minocycline groups.

Spinal microglial activation variations during different stages of CFA-induced inflammatory pain

Following intra plantar injection of CFA, expression of Iba1, as a microglial marker at the lumbar spinal cord, revealed a significant increase on 7th day of the study compared with day zero (p < 0.001). There was significant difference in the Iba1 expression level between day 21 compared with days 0 (p < 0.05) and 7 (p < 0.001) in the CFA group. As shown in Figure 6, administration of Noopept in the CFA + Noopept group considerably decreased the Iba1 expression level on days 7 (p < 0.001) and 21 (p < 0.05) in comparison with the same days in the CFA group. Furthermore, our findings showed that the decrease in spinal Iba1/β-actin expression level in the CFA + Noopept group was significantly greater than what was observed in the CFA + Minocycline group on 7th day compared with the CFA group (p < 0.001). The CFA + Saline (vehicle group) indicated no significant differences in spinal Iba1 expression in comparison with the CFA group at all times of the study (Figure 6).

Figure 6

A: Immunoblots of spinal Iba1 expression in different experimental groups (See also Supplementary Figures 3, 5). All densitometry data were demonstrated as Iba1/ß-actin ratio. B: Long term Noopept administration could change spinal Iba1 expression on days 7 and 21 of the study. Data is presented as mean ± SEM (n = 3/subgroup). ∗P < 0.05 and ∗∗∗P < 0.001 for comparing 21st and 7th days of the study with day 0 in the CFA group. †††P < 0.001 for comparing between days 7 and 21 in the CFA group. #P < 0.05 and ###P < 0.001 for comparing between CFA and CFA + Noopept groups. $$$P < 0.001 for comparing between CFA + Noopept and CFA + Minocycline groups.

Measurement of spinal apoptotic cells during different stages of CFA-induced inflammatory pain

After CFA injection, the apoptosis index significantly increased in the superficial laminas of the ipsilateral lumbar segment of the spinal cord on day 7 (p < 0.001) in the CFA group compared with day zero. In addition, There was significant decrease in the apoptosis index on day 21 compared with day 0 (p < 0.01). Regarding our findings, the number of apoptotic cells considerably decreased on the 7th (p < 0.001) and 21st (p < 0.01) days in the CFA + Noopept group compared with the same days in the CFA group. As presented in Figure 7, There was significant decrease in the percentage of apoptotic cells on the 7th (p < 0.05) and 21st (p < 0.05) days in the CFA + Noopept compared with the same days in the CFA + Minocycline group. There was no significant difference in the apoptosis index in the CFA + Saline group in comparison with the CFA group at all times of the study (Figure 7).

Figure 7

A: Effect of Noopept administration on lumbar spinal cord tissue apoptosis during different phases of inflammatory pain. Arrowheads indicate positive cells. B: Treatment with Noopept following CFA injection could decrease the percentage of TUNEL positive cells in the CFA + Noopept group compared with CFA group. Five non-overlapping zones were randomly selected. The positive cells were counted and an apoptotic index (AI) was calculated depend on following formula: AI (%) = number of TUNEL-positive cells/total number of cells 100. Data is presented as mean ± SEM (n = 3 rats/subgroup, per each time points). ∗∗∗P < 0.001 for comparing between days 0 and 7 in the CFA group. ††P < 0.01 for comparing between days 7 and 21 in the CFA group. ##P < 0.01 and ###P < 0.001 for comparing between CFA and CFA + Noopept groups. $P < 0.05 for comparing between CFA + Noopept and CFA + Minocycline groups.

Cleaved spinal caspase-3 variation during different phases of CFA-induced inflammatory pain

Our results revealed that CFA injection caused a significant increase in cleaved caspase-3 levels on day 7 (p < 0.001) compared with day 0 in the CFA group. There was also significant decrease in cleaved caspase-3 levels on 21st day of study compared with day 7 (p < 0.001) in the CFA group. Long-term Noopept treatment in the CFA + Noopept group significantly decreased CFA-induced caspase-3 activity on the 7th day of study (p < 0.05) compared with the same days in the CFA group. Based on our results, administration of Noopept in the CFA + Noopept group more significantly decreased cleaved caspase-3 levels on both the 7th (p < 0.05) and 21st (p < 0.05) days when compared with the same day in the CFA + Minocycline group. The CFA + Saline group presented no significant differences in spinal cleaved caspase-3 levels in comparison with the CFA group in all time points of the study (Figure 8).

Figure 8

A: Immunoblots of cleavage of spinal caspase-3 in different experimental groups (See also Supplementary Figures 4,5). All densitometry data were demonstrated as Caspase-3/ß-actin ratio. B: Noopept administration reduced cleavage of spinal caspase-3 following CFA-induced inflammation. Data is presented as mean ± SEM (n = 3/subgroup). ∗∗∗P < 0.001: for comparing between days 0 and 7 in the CFA group. †††P < 0.001 for comparing between days 7 and 21 in the CFA group. ###P < 0.001 for comparing between CFA and CFA + Noopept groups. $P < 0.05 for comparing between CFA + Noopept and CFA + Minocycline groups.

Discussion

The current study was designed to clarify the involvement of microglia dependent BDNF in the alteration of pain behaviors and spinal cell apoptosis. We also examined microglial activity and the spinal level of BDNF in Noopept-treated rats in the context of inflammatory pain and spinal cell apoptosis. Our findings in the first step of the study indicated that microglia dependent BDNF and pro-BDNF expressions partially aligned with thermal hyperalgesia, and spinal dorsal horn cell apoptosis increased in the CFA injected rats on day 7 but decreased on day 21. Thus, inhibition of microglial activity by minocycline caused a decrement of these variations during different phases of the study.

Long-term administration of Noopept during persistent peripheral inflammation not only decreases the activity of spinal microglia but also decreases microglia dependent BDNF and pro-BDNF expressions in parallel with thermal hyperalgesia and dorsal horn cell apoptosis. Despite the many studies were done on the effects of Nootropics, such as Noopept, on cognitive disorders like Attention-Deficit/Hyperactivity Disorder (ADHD), Alzheimer's and Parkinson's diseases [66, 67], the mechanism of the effect of this novel dipeptide on persistent inflammatory pain and its involved central mechanisms remained largely uncharacterized. Our results demonstrated that Noopept treatment was not only effective in reducing pain behaviors but also reduced spinal cell apoptosis, which is related to pain behavior variations in the context of CFA-induced inflammation. Apoptosis is a crucial physiological process and any defect in apoptotic mechanisms is closely related to different disorders like pain [63, 64]. This study results showed that the number of apoptotic cells that aligned with the spinal cleaved caspase-3 level during the first week after CFA injection significantly increased in the ipsilateral dorsal horn of the spinal cord in comparison to day zero, which shows the changes in apoptosis following CFA injection were also well related to hyperalgesia during various time points of peripheral inflammation [65]. A large body of evidence exists to substantiate that increment of pain sensitivity is initially due to neural function variations, and microglia has a pivotal role in the pathogenesis of pain [13, 44]. In line with the results from our previous studies, this study shows that CFA-induced inflammation caused persistent spinal microglial activation, which occurred in parallel with hyperalgesia increment, and can be inhibited by Minocycline as a specific microglia inhibitor [31, 45]. It is therefore conceivable that microglia has a vitally important role in the initiation and maintenance of pain, but little attention has been paid to the specific mechanisms of spinal glia cells and their roles in the induction and development of pain behaviors [46, 47]. It is currently well established that, except for certain neurons, activated microglia can release various mediators such as BDNF in response to homeostatic changes [48, 49]. Ulmann et al. demonstrated that following peripheral nerve injury in the context of in vivo and in vitro models, the stimulation of purinergic receptor P2X leads to the release of BDNF from activated microglia [50]. In this study, we found that microglia dependent BDNF and pro-BDNF expressions in the lumbar part of the spinal cord increased on day 7, but decreased on day 21. These results matched with the behavioral outcomes. It is intriguing to note that inhibition of microglia led to a decrement of spinal BDNF and pro-BDNF expression on day 7; however, its effects on BDNF and its precursor on day 21 was not significant. BDNF, a member of the neurotrophin family, is produced following intra and extracellular processing of its precursor, pro-BDNF [15, 51]. Many studies have reported that pro-BDNF is not an inactive biological molecule and has diverse cellular functions [52, 53]. Numerous data convincingly support the notion that the effects of BDNF and pro-BDNF are, in fact, opposite [21]. It is widely accepted that BDNF promotes neuronal survival through activation of the TrKB receptor, while pro-BDNF induces cell apoptosis through p75 receptor activation [54]. However, there is a dearth of information about the relationship of microglia dependent BDNF and pro-BDNF on the spinal dorsal horn in pain behaviors during inflammatory pain. BDNF primarily synthesized as a larger precursor, pro-BDNF, which undergoes proteolytic processing by proteases [55, 56]. It is noteworthy that the conversion of pro-BDNF to BDNF depends on neuronal activity [57]. Matsumoto et al. have shown that the capacity of a neuron for pro-BDNF processing is limited [58]. In this regard, Wysokiński and Mohammadi et al. have reported that a disturbance in BDNF and pro-BDNF balances may be involved in the pathogenesis of mental disorders [59, 60]. Moreover, in vitro studies have indicated that pro-BDNF enhances apoptosis of basal forebrain neurons and sympathetic neurons [61, 62]. The above finding was consistent with changes in microglia dependent BDNF and pro-BDNF expression during the study. So, it seems that microglial BDNF and pro-BDNF imbalances during different phases of the study, specifically on day 7, may lead to apoptosis more than neuronal survival. Therefore, a greater understanding of the role of the microglia signaling pathways in spinal apoptosis and their interaction with hyperalgesia variations opens up the intriguing possibility of manipulating microglial cells to eliminate or reduce pain. It is of interest to note that during this study the administration of Noopept not only reduced spinal Iba1 level, as a microglial activity marker, but also reduced microglia dependent BDNF and pro-BDNF expression levels in the lumbar part of the spinal cord. Although there are considerable studies on the anti-inflammatory effect of Noopept [28, 66], the current study stated the novel dipeptide anti-hyperalgesic and anti-inflammatory effects were mediated via inhibition of microglial activity and apoptosis. Ostrovskaya et al. showed that Noopept, through normalizing incretin system parameters, had an anti-apoptotic effect on pancreatic β cells in the context of experimental Diabetes [68], and in another study, they indicated neuroprotective effects [69]. Moreover, the effects reported in this paper in response to Noopept were significantly more effective than those reported for Minocycline administration in the CFA-inflamed groups, which indicates that there are other pathways involved in Noopept anti-inflammatory effects which need to be investigated. The results of the present experiment and similar reports dealing with experimental models of inflammatory pain suggest that alleviation of pain symptoms and cell apoptosis associated with long-term Noopept treatment may have been mediated more by decreasing the levels of microglia dependent BDNF than pro-BDNF expression. Data obtained from different part of this study did not rule out the involvement of other pathways, specifically in glia cells.

The current study was designed to clarify the involvement of microglia dependent BDNF in the alteration of pain behaviors and spinal cell apoptosis. We also examined microglial activity and the spinal level of BDNF in Noopept-treated rats in the context of inflammatory pain and spinal cell apoptosis. Our findings in the first step of the study indicated that microglia dependent BDNF and pro-BDNF expressions partially aligned with thermal hyperalgesia, and spinal dorsal horn cell apoptosis increased in the CFA injected rats on day 7 but decreased on day 21. Thus, inhibition of microglial activity by minocycline caused a decrement of these variations during different phases of the study.

Conclusion

The results of this study highlight that an alteration of spinal cell apoptosis and thermal hyperalgesia was linked with the amplification of microglial activity and increment of microglia dependent levels of BDNF and pro-BDNF. Furthermore, the anti-apoptotic and analgesic effects of Noopept may be mediated via decreasing microglial activity and microglia dependent levels of BDNF and pro-BDNF during CFA-induced persistent inflammatory pain. In our opinion, nootrophic peptides administration, such as Noopept, can be considered as a new and important target for inflammatory pain reduction, although this conclusion requires further in-depth research.

Declarations

Author contribution statement

Mona Taghizadeh: Performed the experiments; Wrote the paper.

Nader Maghsoudi, Valery Akparov: Contributed reagents, materials, analysis tools or data.

Homa Manaheji, Jalal Zaringhalam: Conceived and designed the experiments.

Mansoureh Baniasadi, Mola Mohammadi, Samira Danyali: Performed the experiments.

Rasoul Ghasemi: Analyzed and interpreted the data.

Funding statement

This work was supported by Research department of the School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran (No:13349).

Data availability statement

Data will be made available on request.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.