Search
Close this search box.

Combined laser-activated SVF and PRP remodeled spinal sclerosis via activation of Olig-2, MBP, and neurotrophic factors and inhibition of BAX and GFAP – Scientific Reports

Study design and population

Fifteen healthy Persian cats, aged 2–3 years, were obtained from pet shops and shelters. The study protocol was reviewed and approved by The Veterinary Medicine Cairo University Institutional Animal Care and Use Committee (Vet-CU-IACUC) under approval number Vet Cu28/04/2021/273. The study was reported in accordance with ARRIVE guidelines.

Each cat was individually housed in suitable cages at the Faculty Animal House, ensuring appropriate room temperature, humidity, and a 12/12 light–dark cycle. Cats were provided with free access to food and water throughout the study. Before spinal cord surgery, all cats were observed for one week to exclude any individuals suffering from preexisting nervous disorders that could confound the study results.

The cats were separated randomly into three groups, with five cats in each. Group I served as the control negative group, consisting of normal, healthy cats. Group II served as the control positive group, where MS was induced using ethidium bromide (EB), followed by intrathecal injection of phosphate-buffered saline through the foramen magnum. Group III, the SVF + PRP group, also underwent MS induction with EB and was subsequently treated with a combination of SVF and PRP 14 days postinduction. The size of the sample in this study was determined based on previous studies conducted in similar experimental setups (Fig. 1).

Figure 1
figure 1

The timeline of treatment.

Surgical procedure and postoperative care for inducing multiple sclerosis

Anesthesia was initiated in feline subjects in Groups II and III with atropine sulfate (0.022 mg/kg) S/C then an intramuscular injection of xylazine (1 mg/kg) (Xyla-Ject® 2% ADWIA Co., A.R.E.) and intravenous administration of ketamine (10 mg/kg) (Ketamar® 5% Sol. Amoun Co. A.R.E). Aseptic preparation of the dorsal area between the neck and hind limbs was conducted. A dorsal midline incision was made along the T12 to L2 vertebrae, followed by dissection of the fascia and severance of the supraspinous ligament around the spinous process. The muscle multifidus lumborum was dissected bluntly to show the dorsal arch of L1. Using a dental drill equipped with a rounded bur with a diameter of 1.2 mm, bilateral holes were created through the dorsal lamina of L1. Each hole received a single injection of 6 μl 0.1% ethidium bromide (EB). The surgical wound was then sutured.

Postoperatively, all cats received fluid supplementation, a course of antibiotics (Ceftriaxone® 25 to 50 mg/kg intramuscularly for 5 days), and analgesics (Meloxicam® EL Nasr Co, A.R. E, 0.2 mg/kg subcutaneously for 3–4 days). Daily clinical assessments were performed on the operated cats, and manual bladder evacuation was conducted twice daily.

Isolation and viability assessment of stromal vascular fraction

Using the same anesthetic protocol, animals in group III were surgically operated as follows: A small skin incision was made in the inguinal region, and approximately 20 g of fat was harvested and placed in a sterile Petri dish. The wound was then sutured.

Under aseptic conditions, the harvested fat was washed three times with PBS and chopped into small pieces. The fat pieces were then transferred to a 50 ml Falcon tube, and an equal volume of collagenase type I (Sigma‒Aldrich, St. Louis, MO, USA) was added for the digestion of extracellular matrices. The tube was incubated at 37 °C for 90 min with continuous agitation. After the digestion period, an equal volume of Dulbecco’s modified Eagle’s medium (DMEM) was added to neutralize the collagenase activity20.

The processed lipoaspirate was centrifuged at 950×g for 10 min to generate a pellet. The pellet was then filtered through a 100 μm nylon mesh and centrifuged again for 10 min at 800×g to eliminate cellular debris. The supernatant was thoroughly aspirated, leaving behind the cell pellets consisting of the stromal vascular fraction. The viability of the cells was assessed using trypan blue dye, and cell counts were performed using a hemocytometer20.

Preparation of platelet-rich plasma (PRP) from whole blood

Under anesthesia, 9 ml of whole blood was drawn from the jugular vein on 1 ml of acid citrate dextrose as an anticoagulant. The first centrifugation step was performed at 450×g for 10 min, resulting in the collection of the buffy coat-containing supernatant plasma. This plasma was then subjected to a second centrifugation step at 850×g for 15 min. Subsequently, the supernatant, containing platelet-poor plasma, was gently aspirated, while the remaining pellet of platelet-rich plasma was resuspended in 0.5 ml of phosphate-buffered saline27,33.

Intrathecal administration of PRP and SVF

The PRP and SVF were combined in a sterile syringe, with the SVF consisting of 10 × 106 nucleated cells and 1.5 × 106 platelets. The mixture was then activated using a red laser diode (635 nm) at a distance of 7 cm for 10 min. In the SVF + PRP group, 1 ml of the activated mixture was intrathecally injected through the foramen magnum. On the other hand, the control-positive group received an intrathecal injection of 1 ml phosphate-buffered saline through the foramen magnum.

Culture and expansion of SVF cells

The SVF pellet was placed in 75 mm3 culture plates containing 15 mL of DMEM high glucose supplemented with 10% fetal bovine serum (FBS) and 100 mg/mL penicillin. The plates were then incubated at 37 °C in a humidified atmosphere with 5% CO2. After 48 h, nonadherent cells were eliminated from the plates, and a fresh medium was added. The medium was changed every 3 days. When the cells reached 80% confluence, they were passaged using a solution of 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA). The cells were incubated with trypsin–EDTA solution for 2 min, neutralized with DMEM, centrifuged, and then replated at a low density for further culture expansion. The process was repeated for 3 successive passages. Once the desired number of cells was achieved, the cells were cryopreserved in liquid nitrogen34.

Gene expression analysis of ADMSC surface markers (CD 44, CD 73, CD 90, CD105)

The easy-spin Total RNA Extraction Kit (iNtRON Biotechnology DR, Cat. No. 17221) was used to isolate the TRNA according to the manufacturer’s instructions. cDNA was generated using M-MuLV Reverse Transcriptase (NEB#M0253) according to the protocol provided35. Real-time reverse transcription (RT)-PCR was used to measure the relative expression of the target genes using the HERAPLUS SYBR Green qPCR kit (#: WF10308002). The primer sets were as follows: CD44: Forward primer: 5’CCCGGGGGCCACTAGCACCTCA-3, Reverse primer: 5’GCCTGGACCACGGGAACCTT-3; CD73: Forward primer: 5’CCCGGGGGCCACTAGCA-CCTCA-3, Reverse primer: 5′GCCTGGACCACGGGA-ACCTT-3 CD90: Forward primer: 5’TCAGGAAA-TGGCTTTTCCCA-3, Reverse primer: 5’TCCTCAATGAGATGCCATAAGCT-3; CD105: Forward primer: 5’CTGGAGCAGGGACGTTGT-3, Reverse primer: 5’GCTCCACGCCTTTGACC-3. The cycle conditions were as follows: 95 °C for 2 min 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Each RT‒PCR was conducted in triplicate. The GAPDH gene was used as an internal control. The data obtained from the qRT-PCR were analyzed using 2- ΔΔCT36.

Differentiation capacity of ADMSCs

When cells reached 70% confluence, cells were trypsinized, and in a six-well plate, 103 cells per well were seeded and cultured in different media to examine differentiation capacity as follows:

Adipogenic differentiation

Adipogenic medium containing Dulbecco’s modified Eagle’s medium with 100 nM dexamethasone, 50 mg/ml indomethacin, and 100 ml ascorbic acid.

Chondrogenic differentiation

Chondrogenic medium containing Dulbecco’s modified Eagle’s medium with 1% fetal bovine serum, 6.25 lg/ml transferrin (Sigma), 10 ng/ml transforming growth factor-b1 (Sigma), and 6.25 lg/ml insulin (Sigma) was used.

Osteogenic differentiation

Osteogenic medium containing Dulbecco’s modified Eagle’s medium with 10% FBS, 0.01 μM dexamethasone, 50 μg/mL ascorbic acid, 10 mM sodium β-glycerophosphate, 10,000 U/mL penicillin, and 10,000 U/mL streptomycin.

The different differentiation media were changed every 3 days for 21 days, and then the cells were washed and fixed in 10% formalin for 20 min and stained with Oil Red O solution in 10% isopropanol, Safranin-O staining, and Alizarin red for 15 min to assess adipogenic, chondrogenic, and osteogenic differentiation, respectively37.

Evaluation of neurological function using the Basso, Beattie, and Bresnahan (BBB)

To assess neurological function, the Basso, Beattie, and Bresnahan (BBB) score38 was calculated at multiple time points posttreatment: 1, 3, 7, 14, 20, and 28 days in all groups. The BBB score ranges from 0 to 21, which mainly evaluates joint function, hind limb coordination, and weight-bearing ability; a higher score indicates higher motor function.

Magnetic resonance imaging (MRI) evaluation of spine

The MRI examinations were conducted using the ECHELON Smart, which is Hitachi’s 1.5 T Supercon MRI system from Japan. The imaging protocol included the acquisition of dorsal T2-weighted, T1-weighted, transverse T2-weighted, T1-weighted, and sagittal short tau inversion recovery (STIR) sequences. The parameters for these sequences, including the repetition time (TR), echo time (TE), and inversion time (TI), were as follows: TR/TE 2880/111 ms for dorsal T2-weighted, TR/TE 623/1 ms for dorsal T1-weighted, TR/TE 3290/99 ms for transverse T2-weighted, TR/TE 651/12 ms for transverse T1-weighted, and TR/TE/TI 3310/61/140 ms for sagittal STIR. The imaging protocol covered the region from the T11 to L3 vertebral bodies20,33.

Macroscopic evaluation

The spinal cords of cats from all groups were exposed and analyzed for morphological characterization from T11 to L3.

Histological analysis of the spinal cord

Spinal cord specimens were obtained and fixed in 10% neutral buffered formalin for 24 h. Formalin-fixed tissue samples of all experimental groups were routinely treated in alcohols and xylenes and then embedded in paraffin wax. Sections of 4–5 μm thickness were prepared for H&E staining39. Subgross-stained sections were obtained using a digital scanning camera (Basler, Germany) mounted to a CX33 light microscope (Olympus, Tokyo, Japan) at 200 X. The stained slides were examined with a light microscope (Leica DM4 B microscope, Germany) and photographed with a digital camera (Leica DMC 4500 digital camera, Germany).

Immunohistochemistry

Unstained tissue sections at 4 µm thickness were obtained on positively charged slides from different groups for IHC staining and quantification. Olig 2, MBP, Bax, and GFAP antibodies were used as previously described in33.

Transmission electron microscopy (TEM)

Samples of the spinal cord were taken and immediately fixed in 3% glutaraldehyde in phosphate buffer for a few hours, postfixed in 1% osmium tetroxide for an hour, and then processed. Later, semithin sections, approximately 1 μm thick, were obtained, stained with toluidine blue, and examined. Finally, ultrathin sections were cut to a thickness of 50 nm and examined at the Electron Microscopy Unit, Faculty of Agriculture, Cairo University (TEM-109, SEO Company).

Quantitative real-time PCR for BDNF, NGF, and SDF genes

Following the manufacturer’s instructions, total RNA was extracted using the RNeasy mini extraction kit (Qiagen, Hilden, Germany). Then, DNase I was used to clean up DNA contamination (Fermentas, Lithuania). Complementary DNA (cDNA) was then produced using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) by the manufacturer’s instructions40. The primer sets for measuring the mRNA levels of target genes was generated using the primer3 tool and was as follows: BDNF: Forward primer: 5’-CGGTCACCGTCCTTGAAAA-3’, Reverse primer: 5’-GGATTGCACTTGGTCTCGTAGAA-3’ NM_001009828.1; NGF: Forward primer: 5’-GCAGGGCAGACCCGCAACAT, Reverse primer: 5’-GCACCACCCGCCTCCAAGTC3’ XM_045033321.1; SDF-1: Forward primer: 5’-ACAGATGTCCTTGCCGATTC-3’, Reverse primer: 5’-CCACTTCAATTTCGGGTCAA-3’ XM_006937984.5; GAPDH: Forward primer: 5’-TGGAAAGCCCATCACCATCT-3’, Reverse primer: 5’-CAACATACTCAGCACCAGCATCA-3’ NM_001009307.1. SYBRTM Green PCR Master Mix was employed in real-time PCR analysis, as previously described, to ascertain the relative expression of the selected genes (Thermo Fisher Scientific, Cat number: 4309155). As directed by the manufacturer, the ABI Prism Step OnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific) was used41. For each sample, the PCRs were run twice. The BDNF, NGF, and SDF expression levels were adjusted to the housekeeping gene (GAPDH). The data obtained from the qRT-PCR were analyzed using 2-ΔΔCT36.

Statistical analysis and data interpretation

This study employed rigorous statistical methods to analyze and interpret gait scores, gene expression data, and immunohistochemistry findings. A two-way analysis of variance (ANOVA) was conducted on randomly selected samples to examine the gait scores. Fisher’s post hoc test was subsequently employed to compare the outcomes of the various treatments. To signify significant differences between groups and experiment days in the columns, letters were utilized. Different superscript letters were used to represent significant differences at a significance level of P ≤ 0.05. The statistical software program Origin Pro, version 2016, was used. Additionally, gene expression analysis and immunohistochemistry data were subjected to one-way ANOVA using GraphPad Prism version 8.4.3 (686). Again, different superscript letters were utilized to indicate significant differences at a significance level of P ≤ 0.0542,43,44.

Ethical approval

All animals were treated and used by following ethical approval from the Veterinary Medicine Cairo University Institutional Animal Care and Use Committee (Vet-CU-IACUC) with approval number Vet Cu28/04/2021/273.

Consent to participate

All the authors have read and approved the manuscript.

Latest Intelligence