Study of the neuroprotective effect of NT-3 dipeptide mimetics GTS-301 and GTS-302 on an experimental model of ischemic stroke

Introduction

Stroke is the second most prevalent non-communicable cause of death and one of the leading causes of disability worldwide. In 2021, about 12 million strokes were registered, more than half of which resulted in death [1]. This grim statistic underscores the relevance of developing new drugs for stroke pharmacotherapy. In this context, great attention is drawn to endogenous proteins of the neurotrophin family (NGF, BDNF, NT-3, NT-4), which possess neuroprotective and neuroregenerative properties [2, 3].

The physiological effects of neurotrophins are mainly mediated by specific tyrosine kinase Trk receptors — TrkA, TrkB, and TrkC [4]. Different neuronal populations express all or only some Trk receptor types, with most neurons expressing at least one of them [5, 6]. Unlike other neurotrophins, NT-3 interacts not only with its cognate TrkC receptors but also with TrkA and TrkB [7], suggesting the potential for neuroprotection in most neuronal populations.

Neuroprotective effects of NT-3 have been demonstrated in an in vivo model of ischemic stroke induced by middle cerebral artery occlusion (MCAO). Zhang et al. [8] showed that intracerebral administration of a viral vector carrying the NT-3 gene 3 days before MCAO reduced brain infarct volume by 40% and improved neurological status from day 1 to day 28 after surgery. Topical application of NT-3 (10 μg in physiological saline) onto the surface of the injured hemisphere during MCAO reduced the volume of ischemic damage by 60% compared to untreated animals and also significantly decreased necrosis and apoptosis in the ischemic area [9]. NT-3 also exhibited neuroregenerative effects in a model of ischemic injury to the sensorimotor cortex (forelimb area) in rats [10]. Administration of NT-3 via a viral vector into the muscles of the injured forelimb 24 hours after ischemia modelling promoted axonal sprouting from corticospinal neurons of the intact hemisphere into the spinal cord, which was accompanied by recovery of sensory and motor functions of the injured forelimb [10]. In vitro studies have shown that inhibition of NT-3 expression in cultured rat cortical neurons under oxygen-glucose deprivation increases their apoptosis [11]. These data further support the neuroprotective properties of NT-3 in cerebral ischemia.

Because the clinical use of neurotrophins is limited by their low bioavailability and the risk of serious adverse effects [12, 13], several research groups are developing low-molecular-weight, pharmacologically suitable mimetics. While some low-molecular-weight mimetics of NGF and BDNF are well characterized and are in preclinical and clinical trials for the treatment of neurological diseases [14], to date there are no reports on the in vivo effects of NT-3 mimetics.

At the Laboratory of Peptide Bioregulators of the Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies, two dimeric dipeptide mimetics of NT-3 — GTS-301 and GTS-302 — were designed and synthesized [15, 16]. In vitro experiments on PC12 cells expressing TrkC receptors showed that GTS-302, like full-length NT-3, activates all major Trk receptor signalling cascades: PI3K/Akt, MAPK/ERK, and PLC-γ1. In contrast, GTS-301 activates only MAPK/ERK and PLC-γ1, with no effect on PI3K/Akt [17].

The aim of the present study was to evaluate the neuroprotective activity of GTS-301 and GTS-302 in a rat model of ischemic stroke induced by MCAO.

Materials and Methods

Chemicals and reagents.
Dipeptides GTS-301 (bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) hexamethylenediamide) and GTS-302 (bis-(N-γ-hydroxybutyryl-L-glutamyl-L-asparagine) hexamethylenediamide) were synthesized at the Laboratory of Peptide Bioregulators, Department of Medicinal Chemistry, Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies, as described previously [15, 16]. Chloral hydrate and formalin were from PanReac Applichem (USA); 2,3,5-triphenyltetrazolium chloride (TTC) from HiMedia Laboratories (India); Tween-80 from ICN Biomedicals (USA).

Animals.
The study was performed on 33 male Wistar rats weighing 230–260 g, obtained from the Andreevka Branch of the Scientific Center for Biomedical Technologies of the Federal Medical Biological Agency of Russia. The animals were kept in a vivarium with free access to food and water under a natural light–dark cycle. Animal experiments were conducted in accordance with Decision No. 81 of the Council of the Eurasian Economic Commission of November 3, 2016 “On the approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in the field of medicinal products circulation”, interstate standards GOST 33215-2014 and GOST 33216-2014 (Guidelines for the care and use of laboratory animals, Appendix A to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS No. 123)). The study was approved by the Biomedical Ethics Committee of the Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies (Protocol No. 2 of January 30, 2023).

Ischemic stroke modelling.
Ischemic stroke was modelled by transient intraluminal MCAO [18]. All surgical procedures were performed using titanium microsurgical instruments. Anaesthesia was induced with a 5% solution of chloral hydrate in physiological saline (350 mg/kg, i.p.). A midline incision was made in the ventral neck region, and the carotid triangle (bounded superiorly by the digastric muscle, laterally by the sternocleidomastoid muscle, and medially by the sternohyoid muscle) was exposed on the right side. The sternohyoid and sternohyoid-sternocleidomastoid muscles were dissected to access the common carotid artery. The vagus nerve was carefully separated from the common carotid artery, and a microvascular clip was temporarily placed 1.5 cm below the carotid bifurcation. The external and internal carotid arteries were carefully dissected from surrounding tissues. The external carotid artery was permanently ligated with a cotton thread 0.5 cm from the bifurcation. The pterygopalatine artery was isolated and coagulated with an electrocautery to prevent excessive bleeding. A temporary vicryl suture was loosely placed around the internal carotid artery, after which the external carotid artery was cut proximal to the cotton ligature. A monofilament (nylon thread 0.25 mm in diameter, coated with silicone for two-thirds of its length) was introduced through the resulting stump. The vicryl loop on the internal carotid artery was loosened, and the monofilament was advanced 19–21 mm from the bifurcation to occlude the middle cerebral artery, then fixed with a microvascular clip. The middle cerebral artery occlusion lasted 60 min, after which the monofilament was carefully and slowly withdrawn, and the incision in the external carotid artery was closed by electrocautery until complete sealing. The internal carotid artery was released from the vicryl suture and the common carotid artery from the microvascular clamp, resulting in full reperfusion.

Sham-operated animals underwent the same surgical procedures except for the transection of the external carotid artery and insertion of the monofilament. The neck incision was closed with cotton thread and treated with streptocide. Body temperature was maintained at 37 °C during the operation and until recovery from anaesthesia using a heating pad.

Rats were randomly divided into four groups: sham operation (Sham, n = 8), MCAO (n = 8), MCAO+GTS-301 (n = 8), and MCAO+GTS-302 (n = 9). GTS-302 was dissolved in water for injection; GTS-301, due to poor water solubility, was suspended in 1% Tween-80 in water for injection. The dose of GTS-302 (1 mg/kg, intraperitoneally (i.p.)) was selected based on previous pharmacological activity studies [12]; GTS-301 was administered at the same dose (1 mg/kg, i.p.). Dipeptides were administered i.p. once daily for 7 days, with the first injection given 4 hours after stroke induction. Rats in the Sham and MCAO groups received water for injection on the same schedule. The injection volume was 2 ml per kg of body weight. Neurological status was assessed on days 3 and 6 after surgery; brain infarct volume was determined on day 7. The study design is shown in Figure 1.

Figure 1. Study design
Note: MCAO — middle cerebral artery occlusion.

Assessment of neurological deficit.
Neurological deficit was evaluated using a modified limb-placing test [19], originally described by De Ryck et al. [20]. This test assesses forelimb and hindlimb responses to tactile and proprioceptive stimulation and consists of 7 different trials for each side of the body. Trials were scored as follows:

• The rat performed the trial completely and without delay — 2 points;

• The rat performed the trial incompletely or with a delay of no more than 2 s — 1 point;

• The rat did not perform the trial — 0 points.

The total score was calculated separately for the left and right sides of the body. The maximum possible score for each side is 14, indicating no neurological impairment.

Determination of brain infarct volume.
Ischemic damage volume was assessed by morphometry of brain sections stained with 2,3,5-triphenyltetrazolium chloride (TTC) [21]. Animals were deeply anaesthetised with 5% chloral hydrate (350 mg/kg, i.p.) and decapitated. The brain was removed and rinsed in physiological saline for 1 min to wash off blood. It was then frozen at –20 °C for 12 min and cut into five 1.2-mm-thick coronal sections using a custom slicing mold and microtome blades. Sections were placed in a Petri dish with 2% TTC in phosphate-buffered saline and incubated for 15 min at 37 °C, then turned over and incubated for another 15 min at 37 °C, followed by fixation in 10% formalin for 30 min. Sections were then placed on slides and scanned on both sides using a flatbed scanner at 2400 dpi in colour mode. Infarct area (unstained region) as well as the areas of the ischemic and intact hemispheres were measured using freely available ImageJ software (National Institutes of Health, USA).

To eliminate measurement errors due to oedema or atrophy in the ischemic hemisphere, the relative infarct volume was calculated using the following formula [22]:

V=Vin×(Vc/Vi)Vc×100%V=Vc​Vin​×(Vc​/Vi​)​×100%

where VinVin​ is the brain infarct volume; ViVi​ is the volume of the ipsilateral hemisphere; and VcVc​ is the volume of the contralateral hemisphere.

Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software, USA). Due to the small sample sizes, non‑parametric statistics were used. Group comparisons were made using the Kruskal–Wallis test followed by Dunn’s post‑hoc test, or the Mann–Whitney U test with Benjamini–Hochberg correction. Differences were considered statistically significant at p < 0.05.

Results and Discussion

During the first 24 hours after surgery, 3 out of 8 animals in the MCAO group, 4 out of 8 in the MCAO+GTS-301 group, and 1 out of 9 in the MCAO+GTS-302 group died; no deaths occurred in the sham-operated group. One rat from the MCAO group was excluded because of unsuccessful stroke modelling (no neurological deficit and no brain infarct).

In the limb-placing test, sham-operated rats showed no neurological deficit on either side of the body: all animals scored 14 points for each side on days 3 and 6 after surgery. In the MCAO, MCAO+GTS-301, and MCAO+GTS-302 groups, the total score for the right (ipsilateral to the injury) side was also 14 on both days, but all animals exhibited neurological deficit on the left (contralateral) side. In the MCAO group, the mean (± SEM) total score for the left side on day 3 was 8±0.7 (p = 0.0008 vs. sham) and on day 6 was 9±0.8 (p = 0.0008 vs. sham) (Fig. 2). Administration of GTS-302 significantly improved neurological status on day 3 after surgery: the score in the MCAO+GTS-302 group was 10.2±0.4 (p = 0.018 vs. MCAO) (Fig. 2). On day 6, the effect of GTS-302 did not reach statistical significance (score 10.9±0.5). GTS-301 showed no statistically significant effect on neurological status.

Figure 2. Neurological status score assessed by the limb-placing test in rats after MCAO
Notes: The total score for the left (contralateral to the injury) side of the body is shown. A — on day 3 after MCAO; B — on day 6 after MCAO. Data are presented as mean values and standard errors of the mean (SEM). *** — p < 0.001 vs. the sham-operated group; * — p < 0.05 vs. the MCAO (vehicle-treated) group (Mann–Whitney U test followed by Benjamini–Hochberg correction).

In the MCAO group, TTC-stained brain sections showed a distinct infarct region (unstained area) involving the cortex and striatum of the right hemisphere (Fig. 3), with an average infarct volume of 34.7±2.8% relative to the intact hemisphere. Administration of GTS-302 significantly (p = 0.0177 vs. MCAO) reduced infarct volume by 39% (to 21.3±2.9%). GTS-301 did not have a statistically significant effect; infarct volume in the MCAO+GTS-301 group was 28.7±5.4% of the intact hemisphere.

Figure 3. GTS-302 dipeptide (1 mg/kg, i.p., 7 days) reduces cerebral infarct volume in a rat model of ischemic stroke induced by middle cerebral artery occlusion (MCAO)
Notes: Infarct volume was calculated relative to the volume of the intact hemisphere, adjusted for potential edema or tissue atrophy in the ischemic hemisphere using the formula described in Methods. Data are presented as mean values and SEM. * — p = 0.048 vs. the MCAO (vehicle-treated) group (Kruskal–Wallis test followed by Dunn’s multiple comparison test).

Thus, the dipeptide mimetic of the fourth loop of NT-3, GTS-302, exhibits neuroprotective activity in the rat MCAO model, reducing brain infarct volume by 39% on day 7 after surgery and improving neurological status on day 3 after surgery. According to the literature [8], intracerebral administration of the NT-3 gene via a viral vector prior to MCAO reduces brain infarct volume by 36% on day 3 and improves neurological status from day 1 to at least day 28 after surgery. Comparison of the effects of GTS-302 with those of full-length NT-3 suggests that the latter has a more pronounced corrective effect on neurological deficits. The similar magnitude of the effect on infarct volume between GTS-302 and NT-3 may be due to the fact that in the case of NT-3, infarct volume was assessed at an earlier time point after MCAO.

The dipeptide GTS-301 was inactive under the studied conditions. This may be because GTS-302 activates all major post‑receptor signalling pathways of Trk receptors — PI3K/Akt, MAPK/ERK, and PLC-γ — whereas GTS-301 activates only MAPK/ERK and PLC-γ [17]. It is well established that the neuroprotective effects of neurotrophins are predominantly mediated by the PI3K/Akt signalling cascade [7]. The MAPK/ERK cascade may also be involved in neuroprotection, but it can also promote neurodegeneration under pathological conditions, including ischaemia [23, 24]. We have previously shown that among dipeptide mimetics of NGF and BDNF, only those compounds that activate PI3K/Akt exhibit neuroprotective activity in the rat MCAO model, with the most active compounds (mimetics of the fourth loops of NGF and BDNF) reducing brain infarct volume by approximately 60% [25]. The results of the present study support the importance of PI3K/Akt activation for the neuroprotective activity of NT-3 mimetics in the MCAO model.

Conclusion

The dipeptide mimetic of the fourth loop of NT-3, GTS-302, which activates the PI3K/Akt cascade (among other pathways), exhibits neuroprotective activity in a rat model of ischemic stroke induced by MCAO. In contrast, GTS-301, which does not activate the PI3K/Akt cascade, is inactive under the same conditions.