Experimental design
Lewis adult female rats (~ 200 g, 7–11 weeks old) were submitted to a unilateral ventral funiculus cut, leading to an intraspinal axotomy of motoneurons on the spinal levels L4, L5, and L6. The animals were divided into the following groups: IA (intramedullary axotomy), IA + DMEM (Dulbecco’s modified Eagle’s medium), IA + FS, IA + MSC, and IA + FS + MSC. Lesion completeness was histologically determined based on the location and extension of the ventral funiculus cut in spinal cord sections stained with cresyl violet (Nissl staining). Motoneurons were identified by the location and size at lamina IX of Rexed, similar to as described in the literature [3, 16, 18, 19, 21,22,23]. Based on the histological evaluation, only animals with complete ventral funiculus cut (reaching the anterior median fissure) and without direct damage to the spinal gray matter were included in the experimental groups. Figure 1c shows the spinal cord histology after a complete unilateral ventral funiculus cut. The contralateral side of the lesion was used as an intern control. For the qPCR experiments, animals without injury were included as a control.
All procedures were carried out with the approval of the Ethics Committee on Animal Experimentation of the University of Campinas, SP, Brazil (CEUA/UNICAMP, protocol 3081-1) and in accordance with the ethical principles regulated by the National Council of Animal Experimentation (CONCEA).
Inflammation and trophic factor production were studied by qPCR (n = 5 in each group) 7 days post injury (dpi), and the production of BDNF by the MSCs was examined by immunohistochemistry (IH) at 14 dpi. Neuronal survival after lesion was evaluated by Nissl staining (n = 5), and synaptic detachment on the surface of axotomized motoneurons and astrogliosis in lamina IX were assessed by IH (n = 5) at 14 dpi. Functional recovery (n = 7), using the CatWalk system, was evaluated at 28 dpi.
Fibrin sealant preparation
The HFS was provided by the Center for the Study of Venoms and Venomous Animals (CEVAP) from São Paulo State University (UNESP), São Paulo, Brazil. Its components and application formula are stated in the patents with record numbers BR1020140114327 and BR1020140114360. The FS polymerizes some seconds after the mixing of its three components: fibrinogen derived from buffalo (Bubalus bubalis) blood, 25 mM calcium chloride, and thrombin-like protein obtained from rattlesnake (Crotalus durissus terrificus) venom [24,25,26].
Mesenchymal stem cell preparation
The MSCs were obtained from the bone marrow of transgenic Lewis rats expressing enhanced green fluorescent protein (EGFP) (LEW-Tg EGFP F455/Rrrc). Homozygous EGFP-positive rats, 4 to 6 weeks old, were euthanized with a lethal dose of halothane. The bone marrow was extracted from the tibiae and femurs and resuspended in DMEM (Dulbecco’s modified Eagle medium—Gibco, Grand Island, NY, USA). The bone marrow cells were plated into 75-cm2 flasks containing DMEM supplemented with 10% FBS (fetal bovine serum—Gibco, Grand Island, NY, USA). Cells were cultured until the fourth passage and applied to animals of the experimental groups IA + MSC and IA + FS + MSC.
MSC phenotyping by flow cytometry
MSCs were characterized by the presence of the following cell surface molecules: CD90, CD54, CD73, and RT1A (MHC I), considered as positive “markers” of these cells [27, 28]. As a negative control, antibodies to CD45 (hematopoietic stem cell marker), CD11b/c (monocyte and macrophage markers), and CD34 (hematopoietic stem cell and endothelial cells marker) were used. After trypsin treatment, approximately 106 cells were incubated for 30 min at 4 °C with the primary antibodies (Additional file 1: Table S1) against CD90, CD54, CD73, RT1A, CD45, CD11b/c, and CD34. Cells were washed with phosphate-buffered saline (PBS) and incubated with Alexa Fluor 488-labeled secondary antibody (Molecular Probes, 1:500) for 30 min at 4 °C. Cells were rewashed in PBS, fixed in 2% formaldehyde, and analyzed on the flow cytometer (Guava® easyCyte ™ 6-2L Flow Cytometer, Millipore, USA). As a negative fluorescence control, the secondary antibody was also added to cells not labeled with the primary antibody. Non-labeled cells were fixed and used to generate the graph of size versus granularity and establish the cell population to be analyzed. The data was analyzed using the FlowJo 7.5.6 software.
Ventral funiculus cut plus heterologous fibrin sealant and mesenchymal stem cell application
The animals were anesthetized with a combination of xylazine (Anasedan®, 10 mg/kg, Sespo Indústria e Comércio Ltda, Paulinia, SP, Brazil) and ketamine (Dopalen®, 90 mg/kg, Sespo Indústria e Comércio Ltda, Paulinia, SP, Brazil). A dorsal incision parallel to the spine was performed at the thoracic region, and the paravertebral muscles were removed. A hemilaminectomy of two vertebrae was performed, exposing the lumbar intumescence. The dura mater was sectioned longitudinally, and the ventral spinal roots L4, L5, and L6 were identified. Using a microscalpel, a unilateral longitudinal incision at the ventral funiculus was made approximately 0.5 mm dorsal to the ventral surface of the spinal cord and extended approximately 2 mm lateral-medial, almost reaching the anterior median fissure. The DMEM, MSCs, and FS were applied directly to the surface of the injured spinal cord, immediately after the lesion, using a micropipette (Fig. 1a, b). The IA + DMEM group received 6 μl of DMEM. For the IA + FS group, the FS was prepared by mixing its three components on the surface of the spinal cord including 3 μl fibrinogen, 2 μl of 25 mM calcium chloride, and 1 μl of thrombin-like protein. For the IA +MSC group, 106 MSCs diluted in DMEM were applied to the spinal cord in a total volume of 6 μl. For the IA + FS + MSC group, 106 MSCs diluted in DMEM in a total volume of 6 μl were mixed with the fibrinogen component of the FS, and the other components were applied as in the IA + FS group. After the surgical procedures, the muscles and skin were sutured in layers, and the animals were kept in the animal house for 7, 14, or 28 days.
Specimen preparation
The animals were anesthetized as previously described and submitted to a thoracotomy. The vascular system was transcardially perfused with cold PBS (pH 7.4). To obtain specimens for RT-qPCR, the left, right, ventral, and dorsal spinal regions were separated. The ventral segment on the ipsilateral side to the lesion was placed in a microtube and snap-frozen in liquid nitrogen. The same area of noninjured spinal cords was used as a control. The specimens were stored in a − 80 °C freezer. For histological analysis, after perfusion with PBS, the animals were perfused with 4% formaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.4. The lesioned region of the spinal cord was dissected, postfixed overnight at 4 °C, and then subjected to gradually increased concentrations of sucrose (10, 20, and 30% for 24 h each). The spinal cords were then embedded into Tissue-Tek O.C.T (Sakura Finetek USA, Inc., Torrance, CA, USA) and frozen at − 35 to − 40 °C. Transverse sections (12 μm thick) were obtained in a cryostat (Micron, HM25, USA) and stored at − 20 °C.
Hematoxylin-eosin and Nissl staining (neuronal survival)
For a histological characterization of the inured anterior funiculus and analysis of inflammatory cell infiltration, hematoxylin-eosin (HE) staining was performed on frozen sections (7 dpi).
Transverse sections of the injured spinal cord (14 dpi) were submitted to Nissl staining (cresyl violet) for neuronal counting after lesion. Motoneurons were identified by their position in the spinal cord (ventral horn, lamina IX), size (substantially larger than interneurons and glial cells), and presence of Nissl substance arranged in polygonal clumps. The motoneurons present in the ventral horn on the ipsilateral and contralateral sides to the injury were counted in alternate sections of each specimen in the injured area of the lumbar intumescence. Only cells with visible nuclei were counted. To correct double counts of neurons, the Abercrombie and Johnson [29] correction formula was used: N = n × t/(t + d), where N is the corrected number of counted neurons, n is the number of counted neurons, t is the thickness of the sections, and d is the mean diameter of the neuron nucleus [29]. The “d” value was calculated based on the average diameter of 15 neuronal nuclei for each experimental group and each spinal side (ipsilateral and contralateral side to lesion).
Immunohistochemistry
The sections were washed with PB and blocked with 3% BSA (bovine serum albumin) in the same buffer for 1 h. The slides were then incubated in a humidified chamber for 3 h with the primary antibodies diluted in 1% BSA and 0.2% Triton in PB. The primary antibodies (Additional file 2: Table S2) used targeted GFAP, synaptophysin, GAP-43, Iba-1, arginase-1, and BDNF. After the sections were washed in PB wash, the secondary antibody CY3 or CY2 (Jackson Immunoresearch, 1:500) according to the affinity to the primary antibody was diluted in 1% BSA and 0.2% Triton in PB and added to the slices, which were then incubated for 45 min in a humidified chamber at room temperature. The slides were washed in PB and mounted on glycerol/PB (3:1).
Immunolabeled slides were observed under a fluorescence microscope (Leica DM5500B). Representative images of lamina IX both ipsi- and contralateral to the lesion were captured per slide (total of three slides per animal, obtained along the injured segments). The quantification of the integrated pixel density, which represents the intensity of protein immunostaining, was performed using ImageJ software (version 1.33u, National Institutes of Health, USA). For the synaptophysin images, eight equidistant areas (80 μm2 each) were measured on the surface of each motor neuron (for three contralateral neurons and three ipsilateral neurons per slide). On the ipsilateral side to the lesion, the synaptic covering was only quantified in neurons positive for GAP-43, indicating that the neuron had been axotomized. For the GFAP images, the integrated density of pixels was measured for the entire image. A percentage ratio between the ipsilateral/contralateral side to the lesion was established. The ratio was calculated for each animal and then for each group ± standard error.
Real-time PCR (RT-qPCR)
The frozen spinal cords were lysed entirely and homogenized in Tryzol (QIAzol Lysis Reagent, Qiagen, Hilden, Germany), and the total RNA was extracted using the kit RNeasy Lipid Tissue Mini Kit (Qiagen), according to the manufacturer’s instructions.
The RNA was eluted in RNase-free water and stored at − 80 °C until the moment of use. The RNA was quantified and analyzed for the absorbance ratios A260/280 nm and A260/230 nm using a nanophotometer. The RNA integrity was evaluated by 1% agarose gel electrophoresis under denaturing conditions. Synthesis of complementary DNA (cDNA) was performed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific/Fermentas) following the manufacturer’s instructions. The cDNA was synthesized with 2 μg of total RNA using oligo (dT)18 as the primer. The cDNA produced was used as template for real-time PCR reactions.
The reactions, always done in triplicate, were carried out using the cDNAs produced, TaqMan®Gene Expression Master Mix (2✕) (Life Technologies/Thermo Fisher Scientific, Carlsbad, CA, USA), RNase-free water, and TaqMan assays (primer + hydrolysis probes) up to a final volume of 20 μL. The assays (Additional file 3: Table S3) used were for GAPDH, HPRT1, iNOS2, Arg-1, TNF-a, IL-1b, IL-6, IL-10, TGF-b, IL-4, IL-13, VEGF, and BDNF. Samples from all animals were tested with two reference genes: GAPDH and HPRT1. The choice of the reference gene was carefully made based on the unchanged expression under all experimental conditions, with HPRT1 shown to be the most adequate. In all cases, negative controls containing RNase-free water instead of the sample were performed. The reactions were performed following the thermocycling indicated by the Master Mix (95 °C for 10 min and 45 cycles of 95 °C for 15 s and 60 °C for 1 min).
Quantitative PCR was performed in the Mx3005P instrument (Agilent, Santa Clara, CA, USA), and the results were calculated using the MxPro software (Agilent). The relative quantification of the genes of interest was calculated using the ΔΔCt method [30].
Functional recovery (CatWalk)
For the gait recovery analysis, the walking track test was performed using the CatWalk System (Noldus Inc., Wageningen, The Netherlands). Two evaluations (three to four runs acquired for each) were performed before the injury. After the injury, evaluations were made daily 3 to 14 dpi and then three times a week until up to 28 dpi for each animal. As no morphological or molecular differences were found between the groups AI and AI + DMEM, the AI + DMEM group was used as the only control. Parameters such as the paw area, stand (duration of the support phase), and swing speed (speed exerted by the paw when it is not in contact with the glass plate) were analyzed by the ratio between the ipsi-/contralateral hind paws. An average was calculated for the preoperative value, each day per animal and each day per experimental group ± standard error. The peroneal functional index (PFI) was calculated using the following formula described by Bain et al. [31]: PFI = 174.9(EPL − NPL/NPL) + 80.3(ETS − NTS/NTS) − 13.4 (E = experimental side; N = normal side; PL = print length; TS = toe spread/paw width) [31].
Statistical analysis
Neuronal survival, immunohistochemistry, and RT-qPCR data were evaluated by one-way ANOVA. CatWalk data were analyzed using the two-way ANOVA method (repeated measures mixed model—ANOVA). To evaluate differences between groups, the Bonferroni post-test was used. For the CatWalk data, after two-way ANOVA, differences between curves/groups were determined by the Mann-Whitney test. The level of significance assumed was equal to *P < 0.05, **P < 0.01, and ***P < 0.001.