Aβ(25–35) neurotoxicity following its injection into the hippocampus was demonstrated by D. R. Rush et al. [3]. Aβ(25–35) injection induced the adjacent tissue loss and neuronal degeneration [3]. However, other authors [4] found no neurotoxic effect of Aβ(25–35) following its administration into the ventral pallidum and substantia innominata. They observed the formation of cavities containing protein aggregates that were positively stained with congo red. Aggregated Aβ(25–35) caused more conspicuous damage of the CA1 pyramidal layer in the hippocampus compared to the peptide synthesized from the same amino acids in the reversed sequence, Aβ(35–25) [5, 6, 7, 8]. Degenerating neurons were also detected in the temporal cortex following the Aβ(25–35) injection into the nucleus basalis magnocellularis of rats [9]. It is important to note that undecapeptide injection into some brain structures can induce transsynaptic cytoskeletal damage and astroglial activation that are observed in the actual injection area and also spread to more distant brain areas. Such changes were detected in the hippocampus following the Aβ(25–35) injection into the amygdala [10]. Our previous work showed that Aβ(25–35) also induced the activation of astrocytes and microglia in the hippocampus after being injected into this structure [8].

Neuroglial activation in the lesion is a controversial phenomenon. On the one hand, being the actual components of neuroinflammation, activated astrocytes and microgliocytes contribute to the degeneration. When triggered, the mechanisms of neuroinflammation can lead to the dysfunction and death of neurons, which exacerbates further inflammation. Thus, the vicious circle is established in which neuroinflammation causes neurodegeneration [11]. On the other hand, glial activation is a distinct compensatory tissue response, with microglia actively phagocyting a pathogen that caused tissue damage, e. g., injected Aβ or amyloid plaque components, and astrocytes contributing to a better supply of neurons with substances necessary for their repair, such as neutrophins.

One of the most important neutrophins of the mammalian brain is a nerve growth factor (NGF). NGF is the main neutrophin that ensures support and functioning of cholinergic neurons in the brain of adult mammals [12, 13]. It is synthesized and released into the extracellular medium by hippocampal and neocortical cells, targets for cholinergic neurons of basal nuclei. In turn, cholinergic neurons in the brain of young, adult and senescing animals express a high affinity NGF receptor (TrkA) and a low affinity NGF receptor (p75NTR) [14], which demonstrates the dependence of the metabolism of those cells on the levels of neutrophins in both the developing and the mature brain. A signal cascade triggered by NGF becomes particularly important in Alzheimer’s development. On early stages of the disease (mild cognitive decline) NGF levels are reduced [15], while later stages (severe dementia) are associated with their increase [15, 16]. Considering a specific role of Aβ at different stages of the disease, it is likely to be involved in NGF metabolism regulation in Alzheimer’s.

The aim of this work was to study the changes in NGF signaling system in the hippocampus of rats following the administration of aggregated Aβ(25–35).

METHODS

Experiments on animals were conducted in compliance with the Directive of the European Parliament and of European Council, dated September 22, 2010, and the Order No 267 of the Ministry of Healthcare of the Russian Federation, dated June 19, 2003, on the protection of animals used for scientific experiments. The protocol of the experiment was approved by the Ethics Committee of the Institute of Higher Nervous Activity and Neurophysiology, RAS.

The study was carried out in male Wistar rats from Stolbovaya breeding nursery of the Medical Center for Biomedical Technologies, FMBA (Moscow oblast, Russia), with weights ranging from 290 to 350 g. The rats were kept in plastic cells in fives under vivarium housing conditions with 12h artificial lighting (8:00 — 20:00) and free access to water and food.

The rats were anaesthetized by an intraperitoneal injection of chloral hydrate (350 mg/kg). Aqueous solutions of Аβ(25–35), a control peptide synthesized from the same amino acids in the reversed sequence Аβ(35–25), and a vehicle (sterile water) were administered bilaterally in the hippocampus at AP –3.8 mm; L ± 2.0 mm; DV +3.8 mm from bregma using Model 900 stereotaxic instrument (David Kopf Instruments, USA) [17]. The rats were injected with 3 nmol aggregated Аβ(25–35) or Аβ(35–25) (Bachem, Switzerland) in a total volume of 2 μL (1.5 nmol/μL), the control group received the equal volume of sterile water. Injections were performed at a rate of 1 μL/min. The needle was left in the injection site for 5 minutes for proper substance distribution and for preventing its leakage. Peptide aggregation was performed as described in [18].

7 days after the surgery the rats were decapitated, their brains removed and washed in ice-cold 0.9 % NaCl solution; hippocampus and cerebral cortex were isolated on ice. Those brain structures were frozen and stored at –85 °С for analysis. To measure the NGF level, the tissue was homogenized at the ratio of 1:10 (mass/volume) in a buffer consisting of 100 mM Tris-HCL (pH 7.0), 2 % bovine serum albumin, 1 M NaCl, 4 mM Na2EDTA, 2 % Triton X-100, 0.1 % NaN3 and protease inhibitors, namely, 157 μg/mL benzamidine, 0.1 μg/mL pepstatin A and 17 μg/mL PMSF.

The total amount of NGF was measured using ChemiKine Nerve Growth Factor Sandwich ELISA Kit, a reagents kit for the enzyme-linked immunosorbent assay (Merck Millipore, USA), according to the manufacturer’s guide. Measurements were performed using Wallac VICTOR 1420 multitasking reader (PerkinElmer, Finland). Protein concentration in the tissue was measured using Coomassie Brilliant Blue G-250 dye. The NGF content was presented in pg/mg protein.

For the histological and enzyme immunoassay, the rats were re-anaesthetized with chloral hydrate (450 mg/kg). Then the brains were fixed by intracardiac perfusion of 4 % paraformaldehyde solution in 0.1 М phosphate buffer (pH 7.4) and stored in the same fixative for 24 hours. 50 μm thick frontal sections were prepared using VT1200 S vibrating microtome (Leica Biosystems, Germany) and stored at –20 °С in a cryoprotectant. The sections were Nissl-stained with cresyl violet (Merck, Germany). The expression of p75NTR receptor was evaluated on free-floating sections by immunohystological assay using polyclonal rabbit antibodies (Sigma-Aldrich, USA) and diluted 1 : 100. Antibody binding was detected using goat-anti-rabbit IgG conjugated with biotin (Sigma-Aldrich, USA) diluted 1 : 800, and VECTASTAIN Elite ABC Kit (Vector Laboratories, USA), an avidin-biotin complex with horseradish peroxidase. Diaminobenzidine (SIGMA Fast kit; Sigma-Aldrich, USA) was used as a chromogen.

A quantitative evaluation of damage degree was performed using the images of Nissl-stained sections taken with Camedia C-4000 (Olympus, Japan). The length of lesions in the dentate gyrus and СА1 hippocampal field was measured using Image-J (NIH, USA) software. Based on the length and thickness of the sections, the total CA1 damaged area was measured as described in our earlier work [19]. Evaluation of p75NTR expression was based on the total area exhibiting positive staining in three sections with the most severe hippocampal damage located 500 μm from each other. To estimate the level of expression in an individual animal, the results were averaged and presented in pixels (pxl).