Effect of a synthetic analogue of thyronamine on the levels of molecular markers of focal brain damage in an experimental model

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Introduction

Ischemic stroke (IS) represents a clinical manifestation of acute focal cerebral ischemia and remains one of the leading global medical and social problems due to its associated high rates of mortality and disability. Stroke is the second most common cause of overall mortality, accounting for 11.8% of deaths [1]. Furthermore, stroke is the third leading cause of permanent disability, leaving 15% to 30% of patients with persistent impairment [2].

Neuroprotection and preservation of the viability of ischemic cerebral tissue in IS are only possible within a narrow therapeutic window. Although reperfusion methods are the gold standard of therapy [3], their application is limited by strict time constraints and potential iatrogenic complications. These limitations necessitate new treatment strategies for both the acute period of IS and its long-term consequences. Promising directions include physical methods (therapeutic hypothermia), innovative pharmaceutical agents, and regenerative medicine approaches using cell technologies [4]. Recent studies have demonstrated potential neuroprotective effects of thyronamines — a class of endogenous derivatives of thyroid hormones (TH) [5]. Intracerebral injections of 3-iodothyronamine (3-T1AM) at submicromolar doses (μg/kg) enhance learning ability, eliminate amnesia, lower pain threshold, and modulate sleep and feeding in mice [6]. Pharmacological administration of 3-T1AM exerts a reversible and dose-dependent effect on body temperature in rodents, inducing hypothermia [7]. This underscores the relevance of studying the potential neuroprotective properties of thyronamines in IS.

The aim of this study was to investigate the effect of a synthetic thyronamine analog (SA-T0AM) on the concentration of key molecular markers of cerebral damage in rat brain tissue in experimental IS.

Materials and methods

Serum biomarkers, while clinically significant [8], do not allow differentiating pathogenetic origin of their elevated concentration —whether it results from activation of specific neuroprotective signaling pathways or from passive diffusion of proteins from damaged neural tissue into the systemic circulation. To address this uncertainty, biomarker monitoring in this study was performed directly in cerebral tissue.

To test the scientific hypothesis regarding the neuroprotective effects of deiodinated thyroid hormone metabolites, we used a water-soluble synthetic analog of thyronamine (4-[4-(2-aminoethoxy)benzyl]aniline, hereinafter referred to as SA-T0AM). The synthesis was performed according to the technology described by G. Chiellini et al. [9] with our own modifications.

The structure of the obtained compound was confirmed by proton nuclear magnetic resonance spectroscopy [10]. Analysis of SA-T0AM by powder X-ray diffraction showed that the studied sample is in a crystalline state. No signs of an amorphous phase were detected. Additionally, an infrared spectroscopic study of the synthetic analog of thyronamine (T0AM) as its dihydrochloride was conducted in the range of 4000–400 cm^–¹ using the KBr pellet method. The spectrum revealed characteristic absorption bands corresponding to the main functional groups of the molecule, including C–H stretch vibrations, deformation vibrations of protonated amino groups, and aromatic ring vibrations. The observed bands confirmed the structure of 4-[4-(2-aminoethoxy)benzyl]aniline dihydrochloride.

The animal study protocol was approved by the Bioethics Committee of the V.K. Gusak Institute of Emergency and Reconstructive Surgery (protocol No. 3 dated November 23, 2023). As an IS model, we used FeCl₃ application to the dura mater [11], as it is simple to perform, characterized by relatively low mortality, and does not require intervention from extraneous factors such as administration of systemic antihypertensive agents or occluders. The model implies a transient nature of ischemia. According to one established concept, FeCl₃-mediated oxidation causes endothelial cell denudation with subsequent exposure of the subendothelial matrix, leading to activation of the coagulation system and subsequent thrombotic occlusion of the middle cerebral artery (MCA). This effect lasts no more than 2 hours after removal of the applications from the meningeal surface, after which blood flow gradually recovers, which most closely resembles the clinical picture in patients with IS [12]. The ischemic focus in the FeCl₃-induced IS model appears to be smaller in volume than in models with proximal MCA occlusion, which explains the better survival rate despite pronounced neurological deficits. Additionally, this model avoids mortality associated with reperfusion after rapid removal of the occluder or clip removal and is not associated with thermal damage to neural tissue, which favorably distinguishes it from the model with distal MCA coagulation.

Ketamine and sibazone (diazepam) were used for anesthesia. After treating the surgical site with an antiseptic solution and subcutaneous bupivacaine injection (0.01 mL), the skin was incised using a transverse approach between the eye and ear, the fascia was separated, and the muscles were transected. A burr at 400 rpm was used to remove a portion of the left temporal bone (under the suture), creating a 3 × 3 mm opening. A sterile gauze pad soaked in 40% FeCl₃ (chemically pure) was applied to the distal portion of the MCA for 5 minutes. After 5 minutes, the application was removed, the site was washed with 0.9% NaCl solution and dried with a cotton swab, the wound was sutured and treated with betadine. The study of the biological SA-T0AM effects was conducted on 30 white laboratory Wistar rats of both sexes, weighing 350–400 g, which were housed under standard vivarium conditions with access to food and water ad libitum. The animals were randomly assigned to groups using a random number method. Apart from randomization, no additional measures were taken to control potential confounding factors. No preliminary inclusion or exclusion criteria were defined for the animals. All animals that underwent the surgical procedure survived to the end of the experiment and were included in the final analysis.

The animals were divided into 4 groups. Rats in the experimental group (n = 12) received an intraperitoneal injection of the study agent at a dose of 50 mg/kg in 1 mL of water for infusion, 60 minutes after the formation of the focal ischemic brain injury. Animals in the control group (n = 6) underwent experimental ischemia without treatment; instead of the agent, they received 1 mL of water for infusion. In the sham-operated group (SO, n = 6), craniotomy was performed, but with application of 0.9% NaCl instead of FeCl₃. Another 6 animals comprised the intact group.

On the 3^rd day after surgery, the animals were euthanized and the brain was extracted. The choice of the study endpoint was determined by the fact that the agent’s effect was evaluated in the acute phase of experimental cerebral ischemia, and this interval corresponds to the phase of early ischemia–reperfusion injury, when neuronal damage, glial activation, and early neuroplasticity develop. For laboratory analysis, the cerebral cortex of rat brains, homogenized in Lysis Buffer-3 (Cloud-Clone Corp.), was used. The homogenate was centrifuged for 5 min at 10,000 g, and the supernatant was aliquoted and stored at –20°C for no more than 5 days until analysis.

To determine brain damage markers, ELISA systems (Cloud-Clone Corp.) designed for use with rat biomaterial were employed. For the study, we selected the following markers: neuroglobin (NGB, pg/mL), glial fibrillary acidic protein (GFAP, pg/mL), brain-derived neurotrophic factor (BDNF, ng/mL), neuron-specific enolase (NSE, ng/mL), and the B subunit of the S100 calcium-binding protein family (S100B, pg/mL) [8, 13, 14]. The analysis was performed in accordance with the manufacturer’s instructions. The data obtained from the analyses were entered into the spreadsheet editor Microsoft Excel 14.0 and subsequently processed using R language v. 4.5.1. After checking the data distribution for normality using the Shapiro–Wilk test, we decided to use parametric statistical methods. To compare two dependent samples (for analyzing differences between the cerebral hemispheres), we used the paired t-test. For correlation analysis, Pearson’s rank correlation coefficient was used. To compare differences between three or more independent samples, one-way analysis of variance (ANOVA) was used. For pairwise post-hoc analysis, the t-test was applied.

Results

Comparison of left and right hemisphere parameters within each group showed no significant interhemispheric differences (Table 1). Mean values of molecular marker levels in the left and right hemisphere cortex are presented in Table 2.

 

Table 1. Results of comparing levels of molecular markers of focal brain injury in the left and right hemispheres (p-value)

Group	Control	Experiment	Sham-operated	Intact
GFAP, pg/mL	0.4468	0.8941	0.6731	0.08223
NSE, ng/mL	0.5851	0.6347	0.6405	0.2444
BDNF, ng/mL	0.4075	0.6588	0.2097	0.1792
NGB, pg/mL	0.3998	0.3994	0.1373	0.2182
S100B, pg/mL	0.1813	0.3414	0.5163	0.3313

 

Table 2. Levels of molecular markers in the cerebral cortex of experimental rats, М ± SD

Marker	Hemisphere	Control	Experiment	Sham-operated	Intact
GFAP, pg/mL	Left	7389.60 ± 541.23	7648.60 ± 886.39	8081.80 ± 337.65	3658.10 ± 210.39
	Right	6890.90 ± 1515.07	7594.70 ± 794.33	8276.44 ± 945.44	2511.70 ± 1648.83
NSE, ng/mL	Left	15.20 ± 10.87	15.10 ± 4.31	15.90 ± 1.16	0.60 ± 0.08
	Right	19.10 ± 6.29	16.10 ± 9.31	17.10 ± 4.70	0.600 ± 0.064
BDNF, ng/mL	Left	242.60 ± 159.31	140.6 ± 70.8	113.50 ± 39.16	37.60 ± 10.82
	Right	180.80 ± 140.81	150.20 ± 66.75	153.10 ± 83.02	47.30 ± 14.99
NGB, pg/mL	Left	1.10 ± 1.26	2.60 ± 1.53	2.80 ± 1.71	0.50 ± 0.51
	Right	0.60 ± 0.12	2.20 ± 1.04	1.80 ± 1.00	0.40 ± 0.36
S100B, pg/mL	Left	390.60 ± 640.73	1284.50 ± 632.84	1371 ± 1016.54	61.20 ± 0.99
	Right	508.90 ± 734.03	1128.90 ± 797.83	1068.50 ± 698.90	84.10 ± 2.40

 

Within groups, a positive correlation was found between the intact and ischemic hemispheres for various molecular markers. In the experimental group, a significant correlation was found for NSE and S100b levels; in the control group, for NGB and S100b (Fig. 1). This suggests that by day 3 post-surgery, the response to ischemia already involves both hemispheres.

 

Fig. 1. Correlation coefficient between marker concentrations in the cortical tissue of the intact and ischemic hemispheres.

* — р < 0.05, ** — р < 0.01.

 

Compared to intact animals, the experimental, control, and sham-operated groups had higher mean levels of GFAP, NSE, and BDNF (Fig. 2), suggesting that these markers are highly sensitive to craniocerebral trauma, and that craniotomy alone leads to an increase in these markers in rat brain tissue.

 

Fig. 2. GFAP, NSE, and BDNF levels in the left and right hemisphere cortical tissue of experimental rats.

 

The mean S100B concentration in both hemispheres was higher in the experimental and sham-operated groups than in the control group and intact animals. However, no statistically significant difference was found between the groups (Fig. 3).

 

Fig. 3. S100B level in the left and right hemisphere cortical tissue of experimental rats.

 

The mean NGB concentration was also higher in the experimental and sham-operated groups. A statistically significant difference (p = 0.03) in NGB levels was found only between the control and experimental groups in the right (non-ischemic) hemisphere (Fig. 4).

 

Fig. 4. NGB level in the left and right hemisphere cortical tissue of experimental rats.

 

Discussion

GFAP is a cytoskeletal component that plays a key role in the reactive astrogliosis of the central nervous system in response to ischemia. As a structural protein, it is not released from cells under physiological conditions and is not detected in the blood of healthy individuals. However, there is reason to suggest that GFAP is associated with intracerebral hemorrhage and traumatic brain injury [15].

NSE is a neuronal cytoplasmic glycolytic enzyme that is found mainly in neurons of the gray matter of the brain and is involved in axonal transport. Elevated NSE levels are observed in response to acute events in the central nervous system, such as cerebral infarction, subarachnoid hemorrhage, traumatic brain injury, hypoxia, and seizures [16–18].

BDNF regulates short-term and long-term synaptic connections, neuroprotection, and modulation of neuronal and glial growth, with the most important role being the control of synapse composition and functionality in various brain regions [19]. At the same time, BDNF plays an important protective role in neuronal survival, structural changes, and plasticity [20].

In our study, an increase in the levels of GFAP, NSE, and BDNF was detected in all 3 experimental groups. We believe that craniotomy itself influences the level of these markers in the cerebral cortex tissue in rats, and therefore they are not indicative for IS models with disruption of the cranium integrity.

S100B is a low-molecular-weight calcium-binding protein that does not cross the blood-brain barrier into the bloodstream under normal conditions and leaks from the cerebrospinal fluid into the peripheral bloodstream when the blood-brain barrier is disrupted. S100B is secreted extracellularly by astrocytes and maintained at low concentrations, thereby promoting synaptic growth, neuronal survival, and to some extent neuroprotection [17]. Conversely, when stimulated by abnormal factors, astrocytes produce and release large amounts of S100B, which exerts neurotoxic effects [21, 22]. In our study, an increase in S100B levels was observed in rats of the experimental and sham-operated groups, but no significant differences between the groups were found. A longer follow-up period for the recovery of central nervous system functions and the engagement of possible compensatory mechanisms may be required.

NGB, a member of the globin protein family, has a high affinity for oxygen and is expressed in brain neurons with a significant degree of specificity. Since NGB affinity for O₂ is similar to that of myoglobin and much higher than that of hemoglobin, it has been hypothesized that NGB may store and transport O₂ in the brain, as myoglobin does in muscles [23]. However, the relatively low NGB concentrations in the brain and its dissociation characteristics make it a less effective O₂ reservoir compared to other respiratory proteins, so its biological function remains unclear.

Most processes that increase NGB levels are directly or indirectly related to disruptions in O₂ metabolism. Given its localization and expression patterns, NGB could potentially serve as a biomarker specific to acute ischemic stroke [8]. However, whether NGB levels increase in hypoxic or ischemic neurons remains controversial [23–25].

A large body of experimental studies indicates a neuroprotective effect of NGB in hypoxic and ischemic injury. NGB expression is associated with improved ischemic injury outcomes and functional regeneration in rodents [15]. Reduced NGB expression increased neuronal hypoxic damage in vitro and ischemic damage in vivo [26, 27]. In transgenic mice, NGB overexpression led to a significant reduction in infarct volume 24 hours after ischemia [28].

NGB exhibits neuroprotective effects by modulating mitochondrial functions, such as adenosine triphosphate production, removal of reactive oxygen and nitrogen species, stimulation of mitochondrial dynamics, and control of apoptosis, as well as by providing protection against neuronal death through G-protein-mediated signaling [29]. NGB exerts a direct antioxidant effect by scavenging not only nitric oxide but also hydrogen peroxide, superoxide anion, hydroxyl radical, and the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt [15]. Possibly, under hypoxic conditions, NGB ensures active transport of O₂ into mitochondria.

On the other hand, there are reports that elevated serum NGB levels after acute IS may be used as a predictor of stroke severity and poor prognosis [30]. In the study by Y. Ramli et al., serum NGB levels in acute IS were higher in patients with unfavorable functional outcomes [8].

Treatment of rats with high doses of triiodothyronine results in NGB overexpression. It is hypothesized that T3 may increase NGB expression by indirectly affecting hypoxia-inducible factor-1 [31].

Currently, it is generally accepted that the primary receptor through which biological effects of thyronamines are mediated, especially in nervous system structures, is TAAR1 [32]. At the plasma membrane, TAAR1 activation leads to its coupling with the Gαs protein and triggers intracellular cAMP accumulation via adenylyl cyclase activation. This induces phosphorylation of protein kinases A and C and upregulation of transcription factors, particularly the cAMP response element-binding protein (CREB) [33]. Thus, an increase in phosphorylation of protein kinases A, B, and CREB was observed in adipocytes exposed to 3-iodothyronamine [34].

CREB is crucial in the neuroprotective transcriptional network [35]. In response to stimuli, CREB can be phosphorylated at the Ser133 site, translocate to cell nuclei, and bind to CREB-responsive elements to activate transcription and expression of multiple antioxidant genes, thereby inhibiting oxidative neuronal damage [36]. These genes include Ngb [37]. It has been reported that in mouse experiments, CREB knockdown significantly reduces NGB protein levels, whereas CREB overexpression significantly increases NGB levels [38]. However, whether the CREB/NGB signaling axis mediates the neuroprotective effects of thyronamines remains an open question.

Our study revealed increased NGB levels after acute focal ischemia modeling surgery in the experimental group and in sham-operated animals, with this elevation reaching statistical significance in the non-ischemic hemisphere of rats receiving SA-T0AM therapy compared to the controls. If we assume that the increase in NGB levels in sham-operated animals is a response to disruption of cranial integrity, then the low NGB levels in the Control group may be associated with depletion of compensatory mechanisms during the IS. Elevated NGB levels in the experimental group may indicate both a positive effect of SA-T0AM therapy on neuroprotection in ischemic stroke and ischemia-independent effects of SA-T0AM. It is likely that the effect of SA-T0AM on NGB levels in intact animals requires further investigation.

We have previously noted that thyronamines may be used in the acute phase of IS to prevent ischemic damage through their thermoregulatory action [39]. A new possible mechanism of their action via an effect on neuroglobin suggests that thyronamines may be effective for ischemic postconditioning in the setting of ischemic damage and prior to reperfusion therapy, with the aim of reducing reperfusion injury, through their modulating effect on mitochondria and influence on oxidative stress.

This study has several limitations. First, direct measurements of the cerebral infarct region were not performed, allowing only indirect assessment of the extent of damage. Second, there was no group of sham-operated animals receiving the study agent, as the study aim was to investigate the neuroprotective effects of a synthetic analogue of thyronamine (SA-T0AM) in IS.

Conclusion

1. On day 3 after modeling cerebral ischemia using the ferric chloride application method, an increased concentration of brain tissue damage biomarkers is observed in the cortex of both hemispheres: the affected and the intact one.
2. When modeling cerebral ischemia using the ferric chloride application method to the meninges, brain tissue damage biomarkers such as GFAP, NSE, and BDNF are of low diagnostic value, as they are nonspecifically elevated in response to the craniotomy procedure.
3. A statistically significant difference was found between the control and experimental groups in the NGB level in the cortex of the right (non-ischemic) hemisphere, which may be related to the ability of SA-T0AM to potentiate compensatory mechanisms in the non-ischemic hemisphere that are activated in response to hypoxia.

About the authors

Dmitry A. Filimonov

V.K. Gusak Institute of Emergency and Reconstructive Surgery

Email: neuro.dnmu@gmail.com
ORCID iD: 0000-0002-4542-6860

Dr. Sci. (Med.), Deputy Director for Research, Head, Department of experimental surgery

Russian Federation, Donetsk

Nadezhda N. Trubnikova

V.K. Gusak Institute of Emergency and Reconstructive Surgery

Email: orenaji3@bk.ru
ORCID iD: 0009-0002-3407-5927

Head, Laboratory of basic research, Department of experimental surgery

Russian Federation, Donetsk

Irina A. Kisilenko

V.K. Gusak Institute of Emergency and Reconstructive Surgery

Email: irinka.dn.15@gmail.com
ORCID iD: 0009-0006-6404-2930

junior research associate, Department of experimental surgery

Russian Federation, Donetsk

Margarita A. Belotserkovskaya

V.K. Gusak Institute of Emergency and Reconstructive Surgery

Author for correspondence.
Email: margarita-amb@mail.ru
ORCID iD: 0009-0004-3019-144X

junior research associate, Department of experimental surgery

Russian Federation, Donetsk

Anzhelika A. Burtseva

V.K. Gusak Institute of Emergency and Reconstructive Surgery

Email: chickis_li@mail.ru
ORCID iD: 0009-0001-6827-6033

laboratory assistant, Department of experimental surgery

Russian Federation, Donetsk

Albina E. Glebova

V.K. Gusak Institute of Emergency and Reconstructive Surgery

Email: aglebova2002a@gmail.com
ORCID iD: 0009-0004-8829-4839

laboratory assistant, Department of experimental surgery

Russian Federation, Donetsk

Roman V. Ishchenko

V.K. Gusak Institute of Emergency and Reconstructive Surgery

Email: ishenkorv@rambler.ru
ORCID iD: 0000-0002-7999-8955

Dr. Sci. (Med.), Director

Russian Federation, Donetsk

Alexander B. Eresko

Joint Institute for Nuclear Research

Email: a_eresko77@jinr.ru
ORCID iD: 0000-0002-3521-5314

Cand. Sci. (Chem.), senior research associate, Laboratory of neutron physics

Russian Federation, Dubna

Elena V. Raksha

Joint Institute for Nuclear Research

Email: a_eresko77@jinr.ru
ORCID iD: 0000-0002-5954-6361

Cand. Sci. (Chem.), senior research associate, Laboratory of neutron physics

Russian Federation, Dubna

Dinara Sh. Diuba

V.K. Gusak Institute of Emergency and Reconstructive Surgery

Email: doctordinara@mail.ru
ORCID iD: 0009-0000-2291-4776

Cand. Sci. (Med.), senior research associate, Academic Secretary

Russian Federation, Donetsk

Elena S. Berdnikovitch

Russian Сenter of Neurology and Neurosciences

Email: berdnickovitch.elena@yandex.ru
ORCID iD: 0000-0002-7608-2255

Cand. Sci. (Ped.), senior research associate, Laboratory for the development of high-tech neurorehabilitation devices, Institute of Neurorehabilitation and Restorative Technologies, Head, Psychology and speech therapy group, Institute of Clinical and Preventive Neurology

Russian Federation, Moscow

References

Supplementary files