Animals and ethics statement

The experimental rats were sourced from the Experimental Animal Center at Shiraz University of Medical Sciences. Fifteen adult male Sprague Dawley rats, aged 6–8 weeks and weighing between 230 and 250 g, were included in the study. Throughout the duration of the investigation, the animals were accommodated in a controlled environment, with a temperature maintained at 23 ± 1 °C, humidity at 50 ± 10%, and a 12:12 light–dark cycle. They had ad libitum access to both food and water. After a seven-day acclimatization period, the animals were divided into three groups.

The study obtained ethical approval from the Local Ethics Committee of Shiraz University of Medical Science (IR.SUMS.REC.1398.1237), supported by Grant Number 18563.

Experimental groups

To assess the relative expression of lncRNA GM12371 at distinct time points post-2-vessel occlusion (2VO) surgery, the experimental groups were categorized as follows:

Sham operation (n = 5)

4 days after chronic cerebral hypoperfusion surgery (2VO-4; n = 5)

28 days after chronic cerebral hypoperfusion (2VO-28; n = 5)

This classification facilitated a comprehensive exploration of lncRNA GM12371 expression during various phases following the induction of chronic cerebral hypoperfusion.

Induction of chronic cerebral hypoperfusion (CCH)

Following anesthesia administration, the ventral area of the animals' necks underwent meticulous cleaning, and a vertical incision was made along the midline. Employing a surgical microscope, subsequent steps included exposing both common carotid arteries, identifying an area devoid of nerves and blood vessels in the carotid sheath, making a gentle incision in this area without causing harm, passing a surgical silk through this small incision, and permanently ligating the common carotid arteries. The sham operation group underwent identical surgical procedures, with the exception of the permanent ligation of the both common carotid arteries. Chronic cerebral hypoperfusion (CCH) induction followed established methods from previous studies [29].

qRT-PCR—quantitative reverse transcription-PCR

We meticulously extracted total RNA from 50 mg of each rat's hippocampus using RNAX Plus (Sinaclon, Tehran, Iran) and subsequently diluted it to a concentration of 500 ng/μl. Their purity was evaluated using a NanoDrop 2000/2000c spectrophotometer (Thermo Scientific, USA). We performed reverse transcription of RNA into cDNA using the Easy cDNA Synthesis kit (Parstous, Mashhad, Iran). The specific primers (Table 1) were designed using AlleleID 7.5 (Premier Biosoft International, Palo Alto, CA) and Generunner. Designed primers were specific exon-exon junctions of genes of interest. For quantitative PCR (qPCR), RealQ Plus 2× Master Mix Green (Ampliqon, Denmark) was utilized in a 10 μl reaction volume, and the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) was employed. The thermocycling conditions involved an initial step at 95 °C for 10 min, followed by 40 cycles of primer-specific temperature for 5 s and 72 °C for 30 s, culminating in a final step of 95 °C for 15 s. Subsequently, a primer-specific temperature was maintained for 1 min, followed by 95 °C for 15 s. The housekeeping gene GAPDH was employed as a control. The relative fold gene expression across distinct experimental groups was calculated using the 2–∆∆Ct method.

Table 1 The sequences of primer used in this study are as followsStatistical analysis

Gene expression results were expressed as Mean ± SEM (standard error of mean). The normal distribution of the data was assessed using the Kolmogorov–Smirnov test. For comparisons, the Kruskal–Wallis test with Dunn's post hoc test was applied. A p-value < 0.05 was considered statistically significant in this analysis. Data organization and analysis were performed using Prism7 and SPSS 20 software.