Pramipexole has a neuroprotective effect in spinal cord injury and upregulates D2 receptor expression in the injured spinal cord tissue in rats

Chemicals and reagents

Pramipexole (HY-17355) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Nissl Staining Solution and ELISA Assay Kit were provided by Beyotime (Shanghai, China). The primary antibodies used in this study were anti-GADPH (ab9485; Abcam, Cambridge, UK), anti-DRD2 (DF10211; Affinity, Shanghai, China), anti-Bax (ab32503; Abcam, Cambridge, UK), anti-NeuN (ab196495; Abcam, Cambridge, UK), anti-Caspase-3 (BS1518; Bioworld, Nanjing, China) and anti-Bcl-2 (ab196495; Abcam, Cambridge, UK). Goat anti-rat IgG (HRP) (SA00001-2; Proteintech, Wuhan Shi, China) was used as the secondary antibody for western blotting.

Animals

Studies involving adult female Sprague-Dawley rats were carried out following the “Guide for the Care and Use of Laboratory Animals”. The research protocol received ethical approval from the Southern Medical University Animal Ethics Committee (approval ID: LAEC-2021-098). Adult female Sprague–Dawley rats weighing 180–200 g were procured from the Experimental Animal Center of Southern Medical University (China). All animals were subsequently raised in a pathogen-free environment with regular light/dark cycles and provided ad libitum access to water and food. The rats were allowed to acclimatize for a week, following which they were randomly assigned to either the control or treatment group.

Establishment of the SCI rat model and PPX treatment

One week after acclimatization, all animals were anaesthetized with an intraperitoneal injection of pentobarbital sodium solution in 0.9% saline (3%, 1.5 mL/kg) 30 min before the surgery. The rats were then secured onto a rat spinal cord adapter (68095; RWD, Guangdong, China). Laminectomy was performed at the tenth thoracic vertebra (T10) using a surgical microscope (SMZ745; Nikon, Minato City, Japan) and a grinding drill (South Korea 90 dental electric grinder), exposing the T10 spinal cord. The T10 spinal cord was fully exposed, revealing a smooth surface and a thick and visible vein in the middle of the back. The spinal cord was impacted by a precision impactor (speed 2.0 m/s, depth 1.5 mm dwell time 0.4 s, 68099II; RWD, Guangdong, China), causing blood vessel bleeding, as well as swelling and congestion of the spinal cord beneath the soft spinal cord, as observed with the surgical microscope. The incision was subsequently sutured in layers using 5-0 threads. After the SCI, the rats’ bladders were manually drained twice daily until the bladder function was regained. The same group of researchers performed all the SCI operation procedures. After the surgery, all rats were administered with an intraperitoneal injection of 1 mL of saline containing 1 × 10⁵ units of penicillin and 1 mg/kg meloxicam for 1 week to prevent infection and dehydration as well as relieve pain.

Female adult Sprague-Dawley rats were divided into four groups (n = 24). (1) Sham group: Rats in this group underwent laminectomy only and were administered 0.9% normal saline solution. (2) SCI group: Rats in this group underwent the SCI procedure described above and received intraperitoneal injections of 0.9% normal saline solution. (3) SCI+PPX (0.25 mg/kg) group: Rats in this group were intravenously injected with 0.25 mg/kg PPX in the tail daily for 28 consecutive days after injury. (4) SCI+PPX (2.0 mg/kg) group: Rats in this group were intravenously injected with 2.0 mg/kg PPX in the tail daily for 28 consecutive days after injury. Pramipexole (HY-17355; MedChemExpress, Monmouth Junction, NJ, USA) was prepared in normal saline at 1 mg/ml.

Basso, Beattie, Bresnahan (BBB) evaluation of locomotion

The movement of both hind limbs of the rats was scored on days 0, 1, 3, 7, 14, 21, and 28 post-injury by two observers blinded to the animal groupings. The BBB evaluation scale ranges from 0 (no observable movement) to 21 (normal locomotion). The rats were placed on a rubber-padded, rectangular hardwood inclined plate with a 5° slope to conduct the evaluations. The inclined plate was then rotated by 5° at a time, and the greatest angle at which the rats could maintain their position for 5 s was recorded. Each animal underwent five evaluations, with the average value as the representative measurement.

RNA preparation and qRT-PCR

Total RNA was extracted from the spinal cord tissue using TRIzol (Invitrogen, Waltham, MA, USA) following the manufacturer’s instructions. The quality of the isolated RNA was evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The extracted RNA was then reverse transcribed into cDNA using the SuperScript™ kit (#10928–034; Invitrogen, Waltham, MA, USA). SYBR Green Pro Taq HS Supermix (AG11701; Accurate Biotechnology, Changsha, China) was used to quantify the mRNA expression level using the Real-Time PCR Detection System (Applied Biosystems in the United States). Each sample underwent at least three independent qRT-PCR amplifications to ensure reliability. Relative gene expression was estimated using the 2^−ΔΔCt method. The primers used for amplification are listed in Table 1.

Western blotting analysis

The spinal cord tissues excised from the experimental animals were lysed in ice-cold RIPA lysis buffer (P0013B; Beyotime, Shanghai, China) supplemented with 100 mM phenylmethylsulfonyl fluoride (PMSF). The tissue was incubated in the lysis buffer at 4 °C for 60 min, followed by centrifugation to extract the supernatant. The protein concentration in the supernatant was determined using the BCA kit (CWBIO, Beijing, China) following the manufacturer’s instructions. The samples were mixed with 5 × loading buffer and heated at 100 °C for 10 min before electrophoresis. The samples were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel (EpiZyme, Shanghai, China) and transferred to nitrocellulose membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 5% skimmed milk for 1 h to prevent non-specific binding. After blocking, the membranes were incubated overnight at 4 °C with specific primary antibodies (bax, bcl-2, caspase-3, DRD2, NeuN, GAPDH) at a dilution ratio of 1:1,000. The next day, the membranes were washed thrice with tris-buffered saline containing 0.1% Tween® 20 detergent (TBST) to remove unbound primary antibodies. Subsequently, the membranes were incubated with appropriate secondary antibodies (goat anti-rabbit, dilution ratio 1:10,000) for 1 h. The ECL detection kit (Bioship, Shanghai, China) was used to visualize the protein bands on the western blot. Subsequently, the images were analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)

The frozen spinal cord tissue was rinsed with pre-cooled 0.1 mol/L phosphate-buffered saline (PBS) to remove residual contaminants. The required tissue sample was weighed, chopped, homogenized (10 mg tissue + 10 uL PBS solution), and centrifuged for 20 min (2,000–3,000 rpm) to obtain a clear supernatant containing the target analyte. The ELISA assay was performed according to the manufacturer’s instructions. The plate (BIO-TEK ELX808; BIO-TEK, El Segundo, CA, USA) was read at the detection wavelength of 450 nm.

Hematoxylin-eosin (H&E) staining and Nissl staining

The rats were anesthetized with 3% pentobarbital sodium, fixed on the operating table, and infused with 200 mL of 0.1 mol/L of PBS and 200 mL of 4% paraformaldehyde (PFA) through the left ventricle. The spinal cord was carefully excised under a microscope and immersed in 4% PFA for 24 h. After fixation, the tissue was dehydrated in an alcohol gradient and then treated with different concentrations of xylene. The tissue samples were embedded into paraffin blocks and sliced into 4 μm thin sections using a paraffin sectioning machine (Thermo Fisher Scientific, Waltham, MA, USA). For Hematoxylin-eosin (H&E) staining, the sections were stained with H&E (Servicebio, Wuhan, China), and the results were observed under a Nikon light microscope (E100; Eclipse, Ottawa, Canada). For Nissl staining, the paraffin sections of tissues were incubated in Nissl Stain at 60 °C for 30 min. After incubation, sections were differentiated with 95% alcohol, dehydrated, and sealed with neutral gum. The surviving neurons in the spinal cord were quantified in three non-overlapping visual fields at 400 × magnification.

Immunohistochemical assay

The spinal cord was carefully removed under a microscope and immersed in 4% PFA for 24 h. After fixation, the tissue was sliced into 4 μm thin sections using a paraffin sectioning machine (Thermo Fisher Scientific, Waltham, MA, USA). The tissue sections were placed in citric acid antigen repair buffer and subjected to microwave heating to facilitate antigen retrieval. The tissue sections were then placed in a 3% hydrogen peroxide solution and incubated in the dark at room temperature for 25 min to block endogenous peroxidase activity. Subsequently, the sections were blocked with 3% bovine serum albumin for 30 min at room temperature. The slices were then incubated with specific primary antibodies (Bcl-2, Bax, and DRD2 at 1:200, NeuN at 1:1,000) overnight at 4 °C. The next day, the tissue sections were incubated with secondary antibodies (horseradish peroxidase-labeled) at room temperature for 50 min. Finally, the tissue sections were stained with 3,3′-Diaminobenzidine, with a brown-yellow color indicating positive results.

Statistical analysis

ImageJ and Prism Version 8.0.2 (GraphPad, San Diego, CA, USA) were used for image processing, while Photoshop (Adobe CS6) was employed to compile the figures. All data are presented as mean ± standard deviation from at least three independent experiments. One-Way ANOVA and Tukey’s multiple comparisons were used for inter-group comparisons, and the least significant difference test was adopted for pairwise comparisons. A P-value < 0.05 was considered statistically significant.

PPX attenuates histological deficits and promotes neurobehavioral functional recovery post-SCI in rats

To assess the neurobehavioral function following SCI, we obtained the BBB and slanting board test scores on days 0, 1, 3, 7, 14, 21, and 28. All model groups exhibited significantly lower scores in both tests compared to the sham group.

At 1 and 3 days after SCI, no significant difference in BBB scores was observed between the groups. However, at 7 days after SCI, the BBB scores of the PPX-2.0 group were significantly higher than that of the SCI group (Fig. 1A). Similarly, at 21 days after SCI, the BBB scores of the PPX-0.25 group were significantly higher than that of the SCI group. No significant differences were observed in the slanting board test scores between the groups during the first 7 days after SCI. Nonetheless, at 14 days post-SCI, the slanting board test scores in the PPX-2.0 group were significantly higher than those in the SCI group (Fig. 1B). Furthermore, at 21 days post-SCI, the slanting board test scores of the PPX-0.25 group were significantly higher than those of the SCI group. These findings suggest the potential of PPX in improving hind limb function recovery.

Furthermore, histological evaluations were conducted through HE and Nissl staining of spinal cord tissues. The sham group exhibited a distinct demarcation between white and gray matter under the microscope, with normal neuron shape, cell density, nucleosome staining, and cytoplasm, and only a minor degree of vacuolar alterations. In contrast, all model groups exhibited a significant decrease in the number of normal neurons 3 days after SCI compared to the sham group, with apparent swelling, pyknosis, and vacuolation in neurons, as well as intense cytoplasmic staining (Fig. 1C). However, by 28 days post-SCI, the pathological changes in the PPX-2.0 group were significantly reduced compared to the SCI group, with increased numbers of normal neurons exhibiting clear nucleosomes, multipolar morphology, and few vacuolar changes (Fig. 1D). Additionally, the SCI group exhibited black and atrophic Nissl bodies 3 days post-SCI (Figs. 1E and 1F). However, in the PPX-0.25 and PPX-2.0 groups, the number of intact neurons increased, though without statistical significance at 3 days post-SCI (Figs. 1E and 1F). Nissl staining indicated that the number of intact neurons in the ventral horn increased in the PPX-2.0 group at 28 days post-SCI compared to the SCI group. On the other hand, the PPX-0.25 group did not exhibit statistical differences at 28 days post-SCI (Figs. 1G and 1H). These results suggest that PPX effectively attenuates histological deficits associated with SCI in rats.

PPX inhibits apoptosis in the spinal cord tissue post-SCI in rats

To study the effect of PPX on apoptosis in injured spinal cord tissue, we employed western blotting to measure the expression levels of Bax/Bcl-2 and caspase-3 proteins in the spinal cord tissue 28 days post-SCI. The SCI group exhibited higher levels of Bax/Bcl-2 and caspase-3 protein expression compared to the sham group, while they were lower in the PPX-0.25 and PPX-2.0 groups compared to the SCI group (Figs. 2A–2C). The mRNA levels of Bax and Bcl-2 in the spinal cord tissue were determined using quantitative RT-PCR 7 days post-SCI. The mRNA levels of pro-apoptotic factor Bax had a greater relative expression in the SCI group than in the sham group; however, Bax mRNA levels were significantly lower in the PPX-2.0 group than in the SCI group (Fig. 2D). Additionally, the relative mRNA level of the anti-apoptotic gene Bcl-2 was higher in both the PPX-0.25 and PPX-2.0 groups compared to the SCI group, while the SCI group exhibited lower Bcl-2 expression compared to the sham group (Fig. 2E). We further employed immunohistochemistry to analyze the expression levels of Bax and Bcl-2 in the spinal cord tissue at various time points after injury in both the SCI and PPX-treated groups. To quantify the expression levels of Bax and Bcl-2, we used a computerized image analysis system to determine the mean optical density (MOD), which correlated with the positive staining intensity of Bax and Bcl-2. We found that the MOD of Bax expression in the spinal cord tissue was significantly higher in the SCI group at 3 days post-SCI compared to the sham group (Figs. 2F and 2H), whereas the MOD of Bcl-2 expression was lower in the SCI group than in the sham group (Figs. 2G and 2I). These findings are consistent with an increase in pro-apoptotic factors and a decrease in anti-apoptotic factors. Notably, at 7 days post-SCI, the PPX-2.0 group exhibited significantly lower MOD values for Bax-positive cells than the SCI group. However, there was no significant difference in Bax-positive cell MOD values between the PPX-0.25 and SCI groups (Figs. 2J and 2L). Furthermore, no significant difference was observed between the PPX-0.25 and SCI groups in the MOD values of Bcl-2-positive cells at 7 days post-SCI (Figs. 2K and 2M). These results collectively suggest that PPX may decrease neuronal apoptosis following SCI and provide neuroprotective effects through the regulation of Bax/Bcl-2 and caspase-3 levels, key regulators of the apoptotic pathway. These findings support the potential of PPX as a therapeutic agent for the treatment of SCI.

PPX inhibits inflammation in the spinal cord tissue after SCI in rats

To elucidate the anti-inflammatory effects of PPX post-SCI, we used ELISA to measure the levels of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) in the spinal cord tissue at 1, 3, and 7 days post-SCI. The SCI group exhibited higher IL-1β and TNF-α protein concentrations than the sham group. In contrast, the PPX-0.25 and PPX-2.0 groups demonstrated lower levels of IL-1β and TNF-α than the SCI group (Figs. 3A and 3B). We also assessed the relative mRNA expression levels of IL-1β and TNF-α in the spinal cord tissue 7 days post-SCI using quantitative RT-PCR. Consistent with the ELISA results, we observed higher relative mRNA expression of IL-1β and TNF-α in the SCI group than in the sham group. Conversely, the PPX-0.25 and PPX-2.0 groups displayed lower relative mRNA expression levels of IL-1β and TNF-α compared to the SCI group (Figs. 3C and 3D). These findings suggest that PPX treatment effectively inhibits inflammation in the spinal cord tissue after SCI.

PPX increases NeuN and DRD2 expression after SCI in rats

Although PPX has demonstrated efficacy in improving hind limb function and promoting recovery after SCI, the underlying mechanism remains elusive. We investigated the change in the expression of DRD2, a protein induced by PPX, in the spinal cord tissue after SCI and PPX administration. Our findings revealed a lower expression of DRD2 in the spinal cord tissue of the SCI group compared to the control group (Figs. 4A and 4B). To assess the impact of PPX on DRD2 expression, we employed western blotting to measure DRD2 and NeuN protein levels in the spinal cord tissue 28 days after SCI. Notably, the PPX-0.25 and PPX-2.0 groups exhibited substantially higher DRD2 expression than the SCI group (Figs. 4C and 4D). While NeuN expression levels between the PPX-0.25 and SCI groups did not differ substantially, the PPX-2.0 group displayed a significantly greater NeuN expression compared to the SCI group (Figs. 4C and 4E). To further validate these findings, we utilized quantitative RT-PCR to analyze the relative expression of DRD2 mRNA in the spinal cord tissue 7 days post-SCI. Although DRD2 mRNA levels exhibited no statistically significant difference between the SCI and PPX-0.25 groups, it was considerably greater in the PPX-2.0 group than in the SCI group (Fig. 4F). Additionally, we observed lower relative expression of DRD2 mRNA in the SCI group than in the sham group. The impact of PPX on NeuN-positive cells in the spinal cord tissue 28 days post-SCI was assessed by immunohistochemistry. Consistent with the western blot findings, although there was no significant difference between the SCI and PPX-0.25 groups, the number of NeuN-labeled cells was significantly higher in the PPX-2.0 group than in the SCI group (Figs. 4G and 4H). Furthermore, we found a significantly lower number of NeuN-positive cells in the spinal cord tissue of the SCI group compared to the sham group. Therefore, our findings demonstrate that PPX treatment could lead to an increase in NeuN expression, which is associated with an increase in DRD2 expression in the spinal cord tissue following SCI.