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

Abstract

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.

Keywords: BDNF; Inflammation; Microglia; Noopept; Programed cell death.

Figure 1

A. Study Groups B. Experimental procedures diagram.

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.

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.

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 7^th 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.

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 21^st and 7^th 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.

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 21^st and 7^th 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.

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.

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.