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Advanced Neurology Exercise modulated Vitamin D and HDL in epilepsy

through PTZ injections using the same protocol as the regions 1.8–3.3 mm posterior to the bregma. Each section,
sham group with a thickness of 8 μm, was meticulously prepared using
(iii) Exercise (EX): Rats underwent exercise 5 days/week a microtome (DS920, Did Sabz, Iran). Ten pairs of sections
for 4 weeks without any injections were selected from each animal and stained with toluidine
(iv) PTZ + EX: Rats received PTZ injections on the same blue. The hippocampal regions and the somatosensory
schedule as the PTZ group and exercised 5 days/week cortex were assessed under light microscopic (C-P8,
for 4 weeks. OPTIKA, Italy). The images were taken using a 40×
2
Chemical kindling through intermittent injection of objective lens. The number of dark cells per 1 mm was
PTZ is a well-established experimental model of epilepsy. counted using the Infinity software (Infinity1, Lumenera,
18
Repeated sub-convulsive injection of PTZ over a month Canada).
induces tonic-clonic seizures resembling those in epileptic 2.5. Statistical analysis
subjects. Accordingly, the PTZ kindling model was
18
employed for this study. PTZ (35 mg/kg) was injected Data were expressed as mean ± standard error of the
every other day for 4 weeks to induce generalized seizures mean (SEM) and analyzed using the one-way analysis
in the rats. of variance and Tukey’s post hoc test. Statistical analyses
were conducted using the Statistical Package for the Social
2.2. Exercise protocol Sciences (SPSS version 23, IBM, United States), with a
In the exercise groups, after acclimation to the treadmill, significance level set at p<0.05.
rats underwent the following protocol: 5 min at 3. Results
10 m/min, 5 min at 15 m/min, 5 min at 20 m/min, and
9 min at 25 m/min. All exercises were performed at 0° 3.1. The serum level of HDL
inclination to maintain moderate intensity. The first and The statistical analysis revealed a significant increase in
the last 3 min of the session served as warm-up and cool- serum level of HDL in the PTZ + EX group compared to
down periods, respectively, at 8 m/min. 19,20 the PTZ and EX groups (Figure 1, p<0.05). The mean ±

2.3. Blood sample collection and biochemical SEM (mg/dL) was 52 ± 0.8 in the sham, 50 ± 0.55 in the
analysis PTZ, 51 ± 0.36 in the EX, and 55 ± 0.7 in the PTZ + EX
groups. There was no significant difference between the EX
Rats were anesthetized through intraperitoneal injection and sham groups.
of a mixture comprising xylazine (5 mg/kg) and
ketamine hydrochloride (50 mg/kg). Blood samples were 3.2. The serum level of LDL
subsequently obtained from the left ventricle using the The statistical analysis indicated no significant differences
cardiac puncture protocol (Paulose and Dakshinamurti, in the level of LDL across all experimental groups. The
1987). The blood samples (2 – 3 mL) were collected and mean ± SEM (mg/dL) was 81.3 ± 0.82, 79.8 ± 0.48, 80.3 ±
centrifuged at 1,500 × g for 10 min at 4°C. The clear serum 0.55, and 81.5 ± 0.63 in the sham, PTZ, EX, and PTZ + EX
was stored at −20°C for the measurement of HDL, LDL, groups, respectively (Figure 2).
and Vitamin D levels. The total serum levels of HDL and
LDL were determined using an enzymatic colorimetric 3.3. The serum level of Vitamin D
method with the commercial DiaSys Kit (Diagnostic The statistical analysis showed that the serum level of
System GmbH, Germany), according to manufacturer’s Vitamin D significantly increased in the EX and PTZ + EX
instruction. groups compared to the PTZ group (Figure 3, p<0.05). The
Total serum level of 25-hydroxy Vitamin D was mean ± SEM (ng/mL) of the sham, PTZ, EX, and PTZ + EX
measured using the Enzyme-Linked Immunosorbent groups were 18.1 ± 0.1, 17.2 ± 0.43, 19.1 ± 0.31, and 19.2
Assay kit (CAT. NO. MBS261766, MyBioSource, United ± 0.18, respectively. There was no significant difference in
States) following the manufacturer’s instructions. the EX and PTZ + EX groups compared to the sham group.

2.4. Tissue preparation and cell counting 3.4. Dark neurons assessment
Saline, followed by 4% paraformaldehyde (PFA), was We calculated the number of damaged neurons in the
perfused transcardially. Subsequently, the brains were hippocampal and cortical areas (Figure 4). The mean
carefully dissected and stored in a solution of 4% PFA number of dark neurons increased significantly in the PTZ
for a week at 4°C. Paraffin-embedded tissue blocks were group compared to the sham group in the cornu ammonis
prepared, and serial coronal sections were obtained from (CA)1, CA3, and cortex areas. Furthermore, the mean

Volume 4 Issue 3 (2025) 80 doi: 10.36922/an.8347