The Impact of Transcranial Direct Stimulation Therapy Combined with Intranasal Near-Infrared Stimulation on Cognitive Performance in Patients with Mild Cognitive Impairment and Alzheimer’s Disease: A Pilot Randomized, Double-Blind, Placebo-Controlled Study

Abstract

Introduction

Alzheimer’s disease (AD) and mild cognitive impairment (MCI) are progressive neurodegenerative conditions with limited therapeutic options. Neuromodulation techniques such as transcranial direct current stimulation (tDCS) and photobiomodulation (PBM) have shown promise in improving cognitive function, but their combined effects remain underexplored.

Methods

In a pilot randomized, double-blind, placebo-controlled trial, 33 participants were assigned to either active or sham stimulation groups. The intervention consisted of 50 sessions over 10 weeks, with tDCS (2 mA, F3–F4 montage) and intranasal near-infrared stimulation (iNIRS) (850 nm, 40 Hz, 50% duty cycle) administered simultaneously. Cognitive outcomes were assessed using the Mini-Mental State Examination (MMSE) and ADAS-Cog at baseline, post-treatment, and at the 12-week follow-up.

Results

Significant improvements were observed in the active group across both scales. MMSE scores increased from 21.89 ± 2.35 to 27.22 ± 1.96 (p = 0.0001), with sustained effects at follow-up. ADAS-Cog scores decreased from 34.78 ± 4.99 to 18.22 ± 4.4 (p < 0.0001). Post hoc analyses revealed significant changes in attention, recall, praxis, and executive domains. No serious adverse events were reported.

Conclusion

Combined tDCS and iNIRS therapy significantly enhances cognitive performance in patients with MCI and mild AD. This synergistic, non-invasive approach may offer a promising therapeutic strategy to delay cognitive decline and reduce care-related burdens.

Trial Registration

ClinicalTrials.gov NCT07290686; Registration date 14 December 2023.

Key Summary Points

Why carry out this study?

Alzheimer’s disease is a progressive neurodegenerative disorder affecting 30 million elderly people worldwide, and the number of patients increases every year. As a result of limited therapeutic options, this remains an unmet need.

Neuromodulation methods such as tDCS and photobiomodulation are promising approaches for improving cognitive function.

This pilot trial aimed to determine whether simultaneous tDCS and intranasal NIRS could synergistically improve cognitive functions in patients with MCI and mild AD.

What was learned from the study?

Combined tDCS and iNIRS significantly improved cognitive performance: MMSE increased from 21.89 ± 2.35 to 27.22 ± 1.96, and ADAS-Cog decreased from 34.78 ± 4.99 to 18.22 ± 4.4, with effects sustained at follow-up.

Combining two neuromodulation techniques may offer a promising therapeutic strategy to delay cognitive decline, but these results should be verified in a larger group in a clinical trial.

Introduction

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder that is responsible for 40–70% cases of dementia among the elderly population. Today, AD affects 30 million elderly people worldwide [1, 2]. AD features a lengthy, asymptomatic preclinical phase that can last about 20 years [3]. As age increases, the number of beta-amyloid deposits rises; however, cognitive reserve and neuroplasticity may delay the transition from asymptomatic to symptomatic disease [4]. Usually, the first clinical sign of dementia caused by AD is impairment of episodic memory (the ability to learn and retain recent information) [5]. Afterward, problems with other cognitive functions develop [6].

Despite the main amyloid hypothesis [6], the exact cause of AD remains unknown. A parallel theory proposes that alterations in specific neural networks and increased cortical excitability are associated with AD [7, 8]. Recent studies show that neuromodulation therapies, such as transcranial direct current stimulation (tDCS) [9–11] and photobiomodulation (PBM), also known as near-infrared stimulation (NIRS) or low-level laser therapy (LLLT) [12–15], have a positive impact on cognitive functions, behavior, and daily activities in patients with AD. In both tDCS and PBM, the target could be areas of the prefrontal cortex. In tDCS, the dorsolateral prefrontal cortex (DLPFC) is targeted, resulting in long-term synaptic potentiation [10, 11]. In the case of NIRS, the target is the default mode network (DMN), which includes the medial prefrontal cortex. The main result of NIR use is the synchronization of connections within the DMN [13, 15]. However, to date, these treatment methods have been used separately in studies.

The aim of the present study was to assess the impact of a therapeutic procedure combining tDCS with intranasal near-infrared stimulation (iNIRS) on cognitive functions in patients diagnosed with mild cognitive impairment (MCI) and AD. Combining these two methods should improve cognitive function in patients with MCI and AD.

Methods

Overview

This placebo-controlled, randomized, double-blinded trial received a positive opinion from the Bioethics Committee at the Medical University of Lodz, No. RNN/336/20/KE, with all subsequent changes approved as KE193/22. The study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki.

The study was conducted in senior day care homes in Lodz between January and April 2024. The subject signed their informed consent up to 3 days before the procedure began. A small group of 33 patients qualified for the pilot study. The planned recruitment target was 50 patients. All inclusion and exclusion criteria are presented in Table 1.

Table 1.

Inclusion and exclusion criteria used in the trial

Inclusion criteria

Exclusion criteria

1. Men and women aged 55–90 2. Patients with MCI and mild AD dementia, diagnosed according to NIA-AA 2011 and DSM-5 criteria 3. MMSE score from 12 to 26 points 4. ADAS-Cog over 17 points 5. Minimum 8 years of education 6. In the case of use of cholinesterase inhibitors and/or memantine, treatment must be at least 3 months before entering the study and at a stable dose for at least 60 days before entering the study

1. Mental retardation 4. Unstable somatic condition 5. Use of benzodiazepines or barbiturates 2 weeks before screening 6. Participation in a clinical trial involving any investigational agent within 6 months before the start of the trial 7. History of epileptic seizures 8. Contraindications to tDCS treatment 9. Patients with depression, bipolar disorder, or psychotic disorder, or any other neurological condition (current or past) 10. Alcoholism or drug addiction as defined by DSM-5 within the last 5 years (addicted for more than 1 year and or in remission for less than 3 years)

AD Alzheimer’s disease, ADAS-Cog Alzheimer’s Disease Assessment Scale-Cognitive Subscale, DSM-5 Diagnostic and Statistical Manual of Mental Disorders, 5th Edn., MCI mild cognitive impairment, MMSE Mini Mental State Examination, NIA-AA National Institute on Aging and Alzheimer’s Association, tDCS transcranial direct current stimulation

Psychiatric and Psychological Procedures

The study population was examined by a psychiatrist using the 2011 National Institute on Aging and Alzheimer’s Association (NIA-AA) clinical guidelines for MCI [4] and AD [5], as well as the DSM-5 criteria, to verify patients’ prior diagnoses of AD or MCI. The patients’ prior neuroimaging studies and medical records, including neuropsychological tests, were also assessed. Geriatric Depression Scale (GDS) and Hachinski Ischaemic Score (HIS) were also used to exclude patients with major depressive disorder and vascular dementia.

Demographic and medical data, including age, sex, education, disease course, comorbidities, medications taken, and smoking status, were also collected.

All patients were evaluated using neuropsychiatric tools, including the Mini-Mental State Examination test (MMSE) and the Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-Cog). Cognitive assessments were performed at baseline, at the end of the procedure (within 3 days of the last treatment session), and at 12-week follow-up.

MMSE is a clinical scale used to examine cognitive dysfunctions in patients. It consists of 30 questions/tasks, allowing for a quantitative assessment of various aspects of cognitive functioning. The areas subject to assessment include orientation in time, orientation in place, memorization, attention and calculation, recalling, naming, repeating, understanding, reading, writing, and drawing.

ADAS-Cog comprises 11 tasks, including both subject-completed tests and observer-based assessments. Together, these tasks assess the cognitive domains of memory, language, and praxis. Specific tasks include word recall, naming objects and fingers, commands, constructional praxis, ideational praxis, orientation, word recognition, and language) [16].

tDCS and iNIRS Protocol

The procedure consisted of daily tDCS sessions lasting 20 min, five consecutive days per week, for 10 weeks. tDCS was delivered at a direct current intensity of 2 mA, with a ramp-up/down time of 30 s, using the anode at point F3 and the cathode at point F4, according to the 10–20 EEG electrode montage system, which covers the left and right dorsolateral frontal cortex, respectively. A pair of 35 cm² electrodes was inserted into sponge pads soaked with 10 mL of sterile water.

During the tDCS procedure, patients received iNIRS via the device, using an IR diode with the following parameters: 850 nm wavelength, 40 Hz pulse frequency, 50% duty cycle, and optical power of 150 mW in a 5-mm transparent package. Stimulation was administered for 20 min, 5 days a week, consecutively. Both stimulations were administered simultaneously.

Sham Protocol

For sham tDCS and iNIRS, the placement of electrodes and diodes and the stimulation parameters were identical to those of the control group; however, the stimulation lasted only 30 s. The duration of the sham procedure was adjusted to mimic the initial sensory experience of tDCS stimulation.

Statistical Analysis

The STATISTICA PL 13.3 statistical package and the R environment (packages: ARTool and lme4) were used for the calculations.

Quantitative variables were described with the mean and standard deviation (SD), and ordinal measures were reported as median (Me) and quartiles (Q25 and Q75), and minimum and maximum (Min–Max). Normality of variables was verified using the Shapiro–Wilk normality test. For categorical variables, the number of observations with a given category was reported. As a result of small sample sizes, percentages were omitted.

The Student’s t test (where the distributions in the compared groups were normal) or the non-parametric Mann–Whitney U test (in the case of non-normality) was used to compare the two independent groups. For qualitative variables, Fisher’s exact test (two-sided) was used to compare groups.

To compare groups with repeated measurements, a classical two-way analysis of variance (AVOVA) with repeated measures was used (in the case of a normal distribution and met assumptions of sphericity (Mauchly test) and equality of variances (Levene’s test)) or, in the case of non-normality, a non-parametric version of the two-way ANOVA—the ART ANOVA (aligned ranks transformation ANOVA). Additionally, a partial eta-squared (η²) value was determined as a measure of effect size. For post hoc comparisons, the Tukey test was applied. For binary variables, generalized linear mixed-effects models of the binomial family were used. In addition, for binary variables, Cochran’s non-parametric Q test was used to test for changes over time (with Dunn’s test for post hoc comparisons).

Results at p < 0.05 were considered statistically significant.

Results

Seventy-four patients were recruited for the study. Of these, 26 were excluded because they did not meet all the inclusion criteria. The most common reason was regular use of benzodiazepines (n = 15). Others had chronic somatic diseases such as epilepsy, hydrocephalus, advanced Parkinson’s disease (n = 4), active depression symptoms, and/or high scores on GDS (n = 6), or were active alcohol users (n = 1). Fifteen patients withdrew consent before (n = 12) or during (n = 3) the stimulation as a result of personal issues (not related to the study). Finally, 33 patients completed the study. To ensure proper blinding, each investigator performed a different procedure. One investigator conducted a diagnostic assessment, assessed patients for inclusion and exclusion criteria, and administered psychological tests prior to stimulation. Then, participants were randomly assigned to either the active group (n = 19) or the placebo group (n = 17). Another researcher performed only the stimulation procedure without knowing which type of stimulation the patient received. The remaining two investigators administered neuropsychological tests once the stimulation procedure was complete; one conducted the assessment immediately after the stimulation ended, and the other at 12 weeks of follow-up.

All patients tolerated tDCS and iNIRS well, with no major adverse effects. Minor side effects were reported by seven patients in the active tDCS/iNIRS group, who experienced itching, tingling, or skin burning under the pads, which resolved within a few minutes during the first two or three sessions. These side effects had no significant impact on participants’ decision to continue in the study. Three subjects declined to participate in the study shortly after randomization because of personal issues. Because they received only one or two stimulations, they were excluded from the statistical analysis. The final number of patients in the treatment group was 18 and that in control group was 15 (Fig. 1). Within the treatment group, there were 8 patients with MCI and 10 patients with AD; the sham group included 8 patients with AD and 7 patients with MCI. The average time from diagnosis to study participation for patients with MCI was 3 years (range 1–6; SD 1.366) and for patients with AD it was 2.25 years (range 0.5–4; SD 1.030). The male-to-female ratios were 2:16 for the active group and 3:12 for the sham group. There were no significant differences between the groups in demographic and baseline data (Tables 2, 3).

Fig. 1.

Study protocol flow diagram. iNIRS intranasal near-infrared stimulation, tDCS transcranial direct current stimulation

Table 2.

Demographic and clinical baseline data

Variable	Group		Group comparisona
	Active (N = 18)	Placebo (N = 15)
Age
Mean ± SD	75.44 ± 6.35	75.8 ± 8.00	t = − 0.1423 df = 31 p = 0.8877
Median [Q1; Q3]	75.5 [71; 80.5]	76 [70.5; 80.5]
Min; Max	65; 86	64; 90
Education in years
Mean ± SD	12.67 ± 2.93	12.83 ± 2.96	U = 131.5 p = 0.9144
Median [Q1; Q3]	12 [10.25; 14]	12 [11; 13.75]
Min; Max	8; 18	10; 21
HIS
Mean ± SD	3.06 ± 1.47	2.67 ± 1.45	U = 112 p = 0.4019
Median [Q1; Q3]	3 [2; 4]	3 [1; 3]
Min; Max	1; 7	1; 6
GDS
Mean ± SD	3.78 ± 1.86	5.2 ± 2.27	U = 85.5 p = 0.0719
Median [Q1; Q3]	4 [2.25; 5]	6 [3; 7]
Min; Max	0; 8	2; 8

HIS Hachinski Ischaemic Score, GDS Geriatric Depression Scale

^aStudent’s t test/Mann–Whitney U test

Table 3.

Demographic and clinical baseline data

Variable	Group		p valuea
	Active (N = 18)	Placebo (N = 15)
Gender
Female	16	12	0.6390
Male	2	3
AChE inhibitors intake
No	15	13	1.0000
Yes	3	2

AChE acetylcholinesterase

^aFisher’s exact test

Two-way ANOVA with repeated measures on the scores for each rating scale, with treatment condition (active and sham tDCS/iNIRS) and time (pre-stimulation; post-stimulation; follow-up) as main factors, revealed significant interactions for both MMSE and ADAS-Cog scale (detailed description below).

Post hoc analysis of MMSE total scores revealed significant improvement in the active group (Fig. 2). Specifically, statistical significance was observed between active total scores pre-stimulation and post-stimulation, with the average MMSE total score increasing from 21.89 ± 2.35 to 27.22 ± 1.96 points (p = 0.0001), as well as between pre-stimulation and follow-up scores in the active group (p = 0.0001). Additionally, a statistically significant difference in follow-up results was observed between the active and placebo groups (p = 0.0002).

Fig. 2.

Effects of transcranial direct current stimulation with intranasal near-infrared stimulation on Mini Mental State Examination

Analysis of ADAS-Cog results revealed significant changes in general scores (Fig. 3). In the active arm, the average score decreased from 34.78 ± 4.99 to 18.22 ± 4.4 (p < 0.0001).

Fig. 3.

Effects of transcranial direct current stimulation with intranasal near-infrared stimulation on ADAS-Cog. ADAS-Cog Alzheimer’s Disease Assessment Scale-Cognitive Subscale

Additionally, a thorough post hoc analysis of the results obtained for the ADAS-Cog scale in individual tasks was conducted. The values in the active group and the placebo group were compared before and after stimulation and at follow-up (interaction group × time).

For each task, statistical significance was achieved for the pre vs. post comparison in the active group (p ≤ 0.0001).

A statistically significant difference was observed across all tasks in the comparison between the active post group and the placebo post group, excluding delayed recall and orientation.

In the word recall task, p = 0.0001 was obtained for the comparison between the pre-active group and the follow-up active group, and p = 0.0346 for the comparison between the post-active group and the follow-up active group.

In the delayed recall task, the comparison between the active group post and the placebo group post did not reveal a statistically significant difference (p = 0.0877), whereas the comparison within the active group from pre to post showed a significant change (p < 0.0001).

Statistical significance was also obtained in all comparisons within the active group (pre vs. post and post vs. follow-up), when comparing the active group post vs. placebo group post (p = 0.0009) and the active group follow-up vs. placebo group follow-up (p < 0.0001) for the construction praxis task.

For the ideational praxis task, statistical significance was demonstrated by comparing the active group’s post-treatment results with those of the placebo group (p = 0.0025) and by comparing the active group’s pre-treatment results with its follow-up results (p < 0.0001).

For the word recognition and deletion tasks, a statistically significant change (p < 0.0001) was obtained in the comparisons within the active group pre vs. post and pre vs. follow-up.

Discussion

In this prospective pilot study, the therapeutic effectiveness of tDCS and PBM was not only confirmed but their synergistic effect was also demonstrated. However, as a result of the small group of patients recruited in the pilot study, the results should be interpreted with caution and verified in a larger group in a clinical trial. According to the post hoc comparisons, patients who received the stimulation showed improved outcomes. Moreover, this effect was maintained over time.

The mechanism of tDCS is to increase neurons’ ability to respond to each subsequent impulse, consequently enhancing excitability through long-term synaptic potentiation [7]. The PBM effect is based on a photochemical mechanism involving the respiratory chain located in mitochondria [17]. The majority of studies related to the usage of PBM and tDCS are aimed at cognitive decline and dementia, especially dementia due to AD. However, these studies assessed the effectiveness of both methods separately.

In the study by Nizamutdinov et al. [15], only transcranial NIRS was applied. They used a wavelength of 1060–1080 nm. The group consisted of 60 patients, with 40 in the active group and 20 in the placebo group. Stimulation was administered over 8 weeks (two sessions per day, each lasting 6 min). Patients performed the stimulation at home, either independently or with the help of a caregiver, which may have affected the quality of the stimulation itself. In our study, stimulation was directly supervised by a physician. Among the neuropsychological scales used—similar to our study—the MMSE was applied, achieving an overall improvement of 4.8 points. Although the effect was significant, its magnitude was smaller than that observed with the combined iNIRS and tDCS therapy. Additionally, the referenced study also used the Clock Drawing Test, Clock Copying Test, LMT-I: Logical Memory Test-Immediate total story unit recall, and LMT-II: Logical Memory Test-Delayed total story unit recall to assess cognitive function, which also yielded statistically significant results, further demonstrating the therapy’s effectiveness. However, these tests were not included in our study.

In another study, Chao [12] applied simultaneous transcranial and intranasal NIRS with a wavelength of 810 nm. Stimulation was performed three times a week over a 12-week period. However, the study involved a small group of patients (n = 8, 4 in the active group and 4 in the placebo group). Despite the small sample size, a reduction of 5.2 points in ADAS-Cog was observed. In our study, the mean total score in the stimulation group dropped by an impressive 16.6 points after just 10 weeks of treatment using iNIRS at 850 nm, combined with tDCS.

In a five-case series report, Saltmarche et al. [18] conducted interventions using intranasal and transcranial PBM with NIR light at a wavelength of 810 nm for both types of stimulation. The intranasal stimulation lasted for 25 min daily over 12 weeks, which is 5 min longer than in our study. However, the transcranial stimulation was only administered in 20-min weekly in-clinic sessions. Specifically, for the first 2 weeks, patients received in-clinic stimulation twice weekly; for the remaining 10 weeks, they attended one session per week. Despite the small sample size, after 12 weeks of active PBM treatment, patients showed significant cognitive improvement: MMSE scores increased by an average of 2.6 points, and ADAS-Cog scores decreased by 6.73 points. Additionally, patients and caregivers reported improvements in sleep, mood, mobility, and overall quality of life, including reduced anxiety, fewer angry outbursts, and better daily functioning—measures not assessed in the current study.

Berman et al. [13] applied transcranial NIRS with a wavelength of 1060–1080 nm over 28 consecutive days, each session lasting 6 min daily. However, the study was conducted on a small group of nine patients, with six participants in the active group and three in the placebo group. Improvement in raw scores was observed in three out of six patients in the active group. As a result of the unrepresentative sample size, statistical analysis was not performed.

In the study by Papi et al. [14], conducted on a group of 42 women with MCI (active group n = 21 and placebo group n = 21), transcranial NIRS with a wavelength of 850 nm was used. Stimulation lasted only 5 days, with each session lasting 10 min, yet a significant improvement in MMSE was observed. In the present study, the same wavelength was used with iNIRS application, but with a substantially higher number of stimulation sessions (50 in total), each lasting 20 min. A comparison of the statistical significance achieved in Papi et al.’s study and ours (p = 0.001 vs. p = 0.0001, respectively) clearly highlights the high effectiveness of NIRS at this wavelength, although longer sessions and a greater total number of sessions are necessary.

Khedr et al. conducted two studies on the effectiveness of tDCS in patients with AD. The first study [11] involved 46 consecutive patients, randomly assigned to two equal groups: active versus sham. Each patient received 10 sessions of anodal tDCS, each lasting 20 min. In the second study [10], 34 patients were divided into three subgroup: anodal tDCS (n = 11), cathodal tDCS (n = 12), and sham (n = 11). Patients in this study also received 10 stimulation sessions, each lasting 25 min. In both studies, significant changes in MMSE were observed in the anodal group, and in the second study, these changes were also observed in the cathodal group, although no differences were found between the two groups.

Experimental studies have shown that anodal stimulation causes neuronal depolarization, leading to increased excitability in patients with AD [19, 20]. For this reason, we only used anodal tDCS in our study.

In available studies, a more detailed analysis of results for individual tasks on both the MMSE and ADAS-Cog scales was also conducted to determine which cognitive functions, in particular, improved with stimulation [21–23].

In the case of the MMSE scale, the results of tasks concerning attention, short-term memory, and praxis (attention and calculation task, delayed recall, following instructions, and constructional praxis) improved.

On the ADAS-Cog scale [24], the most significant improvement was observed in tasks such as following instructions, drawing geometric figures, ideation, and crossing out, which correspond to cognitive domains including praxis, attention, and the ability to maintain concentration. Progressive change was also noticeable in tasks related to short-term memory, such as word recall, delayed recall, and word recognition. The least significant change occurred in tasks related to language functions.

In each of the aforementioned studies, patients reported no serious adverse effects. In the study by Khedr et al., only two individuals reported tingling and itching during the initial tDCS stimulation, which we also observed in our study among seven participants.

Study Limitations

The main limitation of the presented study is the small patient group. Difficulties in recruiting a sufficiently large cohort resulted from comorbidities that precluded participation in the study, as well as frequent dropouts due to low awareness and dissemination of knowledge about non-invasive stimulation methods.

Women appear to be overrepresented in this study. However, data from the Central Statistical Office [25] for Łódź province show that the number of women aged 55 and older exceeds the number of men, and this difference grows with age, reaching a ratio of 2:1 women to men in the group aged 70 and above. Another key factor contributing to the high feminization of this age group is the vibrant textile industry in Łódź during the 1970s and 1980s, which primarily employed women as textile workers. As a result, women have a significant participation in our clinical trial.

Additionally, some tests may lack the power to detect smaller changes. As a result, large improvements in some individuals might be hidden by minimal changes in others, and vice versa, especially when analyzing pooled data from patients at different stages of the disease. One way to address this problem is to group patient data by dementia severity.

Conclusions

A total of 50 sessions of tDCS stimulation combined with iNIRS can significantly enhance cognitive functions in patients with MCI and mild AD. It may also decrease the social exclusion faced by many patients with dementia and reduce the social costs associated with intensive care requirements. However, as a result of the small group of patients recruited in the pilot study, the results should be verified in a larger group in a clinical trial.

Authors’ Contributions

Conceptualization: Joanna Woźniak-Mitał, Jakub Kaźmierski; data curation: Joanna Woźniak-Mitał, Marta Strombek-Milczarek, Paweł Rasmus; formal analysis: Joanna Woźniak- Mitał, Jakub Kaźmierski; investigation: Joanna Woźniak-Mitał, Jakub Kaźmierski, Marta Strombek-Milczarek, Paweł Rasmus; methodology: Joanna Woźniak -Mitał, Jakub Kaźmierski, Marta Strombek-Milczarek, Paweł Rasmus; writing- original draft: Joanna Woźniak-Mitał; writing—review and editing: Jakub Kaźmierski, Marta Strombek-Milczarek, Paweł Rasmus. All authors read and approved the final manuscript.

Funding

No funding or sponsorship was received for this study or publication of this article. The Rapid Service Fee was funded by the authors.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of Interest

Joanna Woźniak-Mitał has nothing to disclose. Paweł Rasmus has nothing to disclose. Marta Strombek-Milczarek has nothing to disclose. Jakub Kaźmierski has nothing to disclose.

Ethical Approval

Study with informed consent were approved by The Bioethics Committee at the Medical University of Lodz, No. RNN/336/20/KE, with all subsequent changes approved as KE193/22. The study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki.