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A pilot study of cell-mediated gene therapy for spinal cord injury in mini pigs
Accepted Manuscript Title: A pilot study of cell-mediated gene therapy for spinal cord injury in mini pigs Authors: Islamov Rustem Robertovich, Sokolov Mikhail Evgenyevich, Bashirov Farid Vagizovich, Fadeev Filip Olegovich, Shmarov Maxim Michaylovich, Naroditskiy Boris Savelyevich, Povysheva Tatyana Vyacheslavovna, Shaymardanova Gulnara Ferdinantovna, Yakupov Radik Albertovich, Chelyshev Yuri Aleksandrovich, Lavrov Igor Aleksandrovich PII: DOI: Reference:
S0304-3940(17)30140-4 http://dx.doi.org/doi:10.1016/j.neulet.2017.02.034 NSL 32646
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
22-6-2016 24-11-2016 12-2-2017
Please cite this article as: Islamov Rustem Robertovich, Sokolov Mikhail Evgenyevich, Bashirov Farid Vagizovich, Fadeev Filip Olegovich, Shmarov Maxim Michaylovich, Naroditskiy Boris Savelyevich, Povysheva Tatyana Vyacheslavovna, Shaymardanova Gulnara Ferdinantovna, Yakupov Radik Albertovich, Chelyshev Yuri Aleksandrovich, Lavrov Igor Aleksandrovich, A pilot study of cellmediated gene therapy for spinal cord injury in mini pigs, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2017.02.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A pilot study of cell-mediated gene therapy for spinal cord injury in mini pigs
Islamov Rustem Robertovich1*, Sokolov Mikhail Evgenyevich1,2, Bashirov Farid Vagizovich1, Fadeev Filip Olegovich1,2, Shmarov Maxim Michaylovich4, Naroditskiy Boris Savelyevich4, Povysheva Tatyana Vyacheslavovna1, Shaymardanova Gulnara Ferdinantovna1, Yakupov Radik Albertovich3, Chelyshev Yuri Aleksandrovich1,2, Lavrov Igor Aleksandrovich2,5*
1
Kazan State Medical University, Kazan, Russia
2
Kazan Federal University, Kazan, Russia
3
Kazan State Medical Academy, Kazan, Russia
4
Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia
5
Departments of Neurologic Surgery and Biomedical Engineering, Mayo Clinic, Rochester, MN,
US *
corresponding authors
*Correspondence (Igor Lavrov, MD, PhD): Address: Department of Neurologic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, US Tel: +(1) 507-538-7400 E-mail: [email protected]
Highlights
Intrathecal injection of the gene-cell constraction (UCBC-based delivery of three therapeutic genes) was used for the first time for SCI treatment in large animal mini-pig SCI model. Results provide evidence of feasibility and potential efficacy of proposed UCBC-based delivery of three therapeutic genes therapy. Recovery of the motor functions, somatosensory evoked potentials, and histological findings in control and treated animals support the positive effect of the gene-cell constriction for recovery after spinal cord injury in large animal model.
Abstract Currently, in clinical practice there is no efficient way to overcome the sequences of neurodegeneration after spinal cord traumatic injury. Using a new experimental model of spinal cord contusion injury on miniature pigs, we proposed to deliver therapeutic genes encoding vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM) to the damaged area, using umbilical cord blood mononuclear cells (UCBC). In this study, genetically engineered UCBC (2×106 cells in 200 µl of saline) were injected intrathecally to mini-pigs 10 days after SCI. Control and experimental mini pigs were observed for 60 days after surgery. Histological, electrophysiological, and clinical evaluation demonstrated significant improvement in animal treated with genetically engineered UCBCs. Difference in recovery of the somatosensory evoked potentials and in histological findings in control and treated animals support the positive effect of the gene-cell constriction for recovery after spinal cord injury. Results of this study suggest that transplantation of UCBCs simultaneously transduced with three recombinant adenoviruses Ad5-VEGF, Ad5-GDNF and Ad5-NCAM represent a novel potentially successful approach for treatment of spinal cord injury.
KEYWORDS: Cell-mediated gene therapy, adenoviral vector, Spinal Cord Injury, Mini pigs, Human umbilical cord blood mononuclear cell, Vascular endothelial growth factor (VEGF), Glial cell-derived neurotrophic factor (GDNF), Neural cell adhesion molecule (NCAM).
Introduction Spinal cord injury (SCI) is classified as a neurotrauma, although it is followed by disturbance in blood supply, neurodegenerative and other pathological consequences [1]. Regardless numerous animal studies and clinical trials, to this time there is no successful clinical protocol for SCI treatment. To enhance the efficacy of SCI therapy along with experiments on rodents and clinical trials, experiments on large animals models appear to be highly important. Most of the previous studies on neuroregeneration were performed on rodents and reported positive results require further investigations on large animal models similar in anatomy and physiology to human [2,3,4]. Between others available experimental models mini pigs are seem to be one of the optimal because of anatomic, physiologic and ethical reasons [2]. The importance of performing experiments on swine is dictated by several reasons, such as (1) anatomical considerations, thus, the length of neuritis in human is more then 100 times longer compare to rodent, (2) sharing with
human common physiological characteristics, (3) pharmaceutical reasons, such the dosage and the way of drug delivery is close to human. These details are vary from model to model and particularly between the small and large animals and the positive results obtained on rodents may not be directly applicable on patients [5,6]. Current progress in gene therapy for spinal cord injury aimed to protect neurons and stimulate the growth of neuritis [7]. Both the direct gene therapy, as well as a cell-mediated gene therapy for spinal cord injury, is actively studied during the last years. The therapeutic genes and types of cells for genes delivery are diverse, although the best gene or gene-cell constriction for stimulation of neuroregeneration is still has to be identified [8]. Up to date a big variety of cell types and viral vectors are employed for cell-based gene delivery into CNS in rodents. Direct gene therapy or cell-based gene delivery following the SCI was successful in preservation of neurons and neuronal fibers and in improving locomotion in rats [9,10,11]. In our previous experiments, using the model of spinal cord injury in rats and transgenic G93A mice with amyotrophic lateral sclerosis (ALS), we successfully developed the gene-cell construction based on umbilical cord blood mononuclear cells (UCBC) and adenoviral vectors expressing neurotrophic factors (VEGF and GDNF) and neural cell adhesion molecule (NCAM) [12]. Adenoviral vector carrying GDNF for direct gene or UCBC-mediated GDNF gene therapy of spinal cord contusion in rat showed advantages of both methods. Co-expression in UCBC of recombinant VEGF and NCAM or GDNF and NCAM also showed symptomatic improvement, increased life span and sustained cell homing after transplantation into ALS mice. Our results suggest that the tandem overexpression of VEGF, GDNF (as a therapeutic molecules) and NCAM (as a molecule for survivability and addressed homing of UCBC) in genetically engineered UCBC can be useful in treatment of SCI in mini pigs. Based on our previous results with UCBC-mediated gene therapy on SCI rat model, we proposed a pilot experiment on mini pigs with genetically engineered UCBC. The goal of this study was to develop and evaluate methodological aspects of a new treatment strategy of UCBS transplantation combined with three therapeutic genes in a large animal model of the mini-pig. To address this goal, we investigated the therapeutic efficacy of UCBC transduced with VEGF, GDNF and NCAM after intrathecal transplantation in mini-pigs with spinal cord contusion.
Methods This study was conducted in compliance with local IACUC protocol (institutional animal protocol at Kazan State Medical University (KSMU), # 5, from May 27, 2014) and IRB protocol based on the license of 'Stem Cell Bank' of KSMU. All pregnant women were signed informed consent for using their umbilical cord blood and were thoroughly examined before the delivery for possible contraindications for umbilical cord blood cell donation. Umbilical cord blood was collected by trained personnel of the maternity hospital according to the instruction of Stem Cell Bank of KSMU. Preparation of genetically modified human umbilical cord blood mononuclear cells Adenoviral vectors generation. The plasmids and the recombinant human adenoviruses of serotype 5 Ad5-VEGF165, Ad5-GDNF, and Ad5-NCAM1 were obtained using VEGF165, GDNF, and NCAM1 genes, respectively. The nucleotide sequences encoding VEGF165 (Gene Bank NM_001171626.1), GDNF (Gene Bank NM_019139.1) and NCAM1 (Gene Bank NM_001076682.2) were obtained by chemical synthesis in “Evrogen” JSC. The AdEasy Adenoviral Vector System was used to construct Ad5-VEGF165, Ad5-GDNF and Ad5-NCAM1 according to the manufacturer’s instructions. rAds were grown in HEK-293 and purified by exclusion chromatography. The titers of Ad5-VEGF165, Ad5-GDNF and Ad5-NCAM1(2×109 PFU/ml, 4×109 PFU/ml and 1×109 pfu/ml, respectively) were determined by the plaque formation technique in the HEK-293 cell culture.
Cell preparation. Umbilical cord blood was obtained from healthy women during normal delivery. The blood was processed in accordance to the protocol of the legitimate and ethical standards generally accepted in the stem cell bank of the Kazan State Medical University. Mononuclear fraction of human UCB was isolated by standard technique of sedimentation on to a density barrier (Ficoll, 1.077g/ml) as described previously [13]. After purification UCBCs were cultivated in RPMI-1640 medium supplemented with 10% FBS and mixture of antibiotics penicillin and streptomycin (100 U/ml, 100 micrograms/ml) (Sigma, USA). UCBC were seeded in 10 cm culture dish and simultaneously transduced with three recombinant adenoviruses Ad5VEGF, Ad5-GDNF and Ad5-NCAM at MOI 10. Afterwards cells were incubated for 12-16 hours in a humid environment at a temperature of +37°C with 5% CO 2 content, washed with DPBS solution and prepared for transplantation to experimental group of animals. Animals and treatments In the study 6-month-old female Vietnamese Pot-Bellied miniature pig weighing 5 kg were employed. For two weeks before the surgery animals were kept solely in a housing area with 12 h light/dark cycles, controlled temperature (24-25°C) and water/food supply. All experimental procedures were performed in accordance with the Kazan State Medical University Animal Care and Use Committee guidelines. Contusion spinal cord injury model. At the day of surgery pre-medication with intramuscular injection of ketamine (5 mg/kg) and diazepam (0.5 mg/kg) was performed. Antibiotic cefazolin (60 mg) was given intramuscular before surgery. Anesthesia was induced with propofol and afterwards animals were endotracheally intubated and maintained on isoflurane (1.3%). Rectal temperature was evaluated continuously during surgery and electrophysiological tests and regulated with thermal blankets. The operative area was cleaned with povidone-iodine, dressed with sterile material. Intravenous infusion and postoperative drug administration was performed via the venous catheter. An urinary catheter was placed during the surgery and was maintained post-operatively. Contusion model with weight-drop device was used in our group over last years and was designed based on spinal cord contusion rodent model described previously [3, 14]. Briefly, for SCI a laminectomy was performed at the Th9–Th10 vertebral level and a custom modified weight-drop device ensured falling of the 50-g weight from the 50 cm height on the spinal cord causing the contusion injury [15]. Cell transplantation procedures. Ten days after surgery saline or UCBC were injected intrathecaly under ketamine (5 mg/kg) and diazepam (0.5 mg/kg) anesthesia at the L4–L5 vertebral level. A control animal received intrathecal injection of saline and an experimental mini pig received 2Ч106 UCBC in 200 µl of saline [12]. Behavioral assessment After animal acclimatization, animals were trained for one hour per day for total five days to walk with body weight support. Locomotor function was determined according to PTIBS (Porcine Thoracic Injury Behavior Scale) [2] and muscle tone in front and rear limbs were evaluated. PTIBS assessment was performed once a week during nine weeks period, when each animal was walking through a narrow 10m long corridor with transparent borders, while recorded on a camera. During the PTIBS assessment visually all steps were evaluated and the inter-rater agreement between 5 raters was quantified with Fleiss' kappa. Signs of pain sensitivity (skin pinch test) estimated by vocal response and muscle tone in forelimbs and hind limbs were evaluated. Somatosensory Evoked Potential (SSEP)
For SSEP assessment we used 2-channel digital miniature electromyograph «Neuro-EMGMicro» (Neurosoft, Russia). The baseline somatosensory evoked potentials (SSEP) were evaluated a week before surgery. After the surgery SSEP assessment was performed 4 times: one day before UCBC transplantation (Phase 1), 3 weeks (Phase 2), 5 weeks (Phase 3), and 7 weeks (Phase 4) post UCBC transplantation. All areas of recording were prepared with rubbing ethanol. Standard electrodes were used for stimulation of the n. suralis and n. medianus. An active recording electrode was placed in projection of the somatosensory cortex on contrаlateral to stimulation side and the reference electrode was placed at the point of intersection of the perpendicular line from the active electrode to the sagittal line of the head. Electrodes were coated with an electroconductive gel and fixed with adhesive tape to the scalp. The stimulating electrodes were placed at the projection of the median nerve at the elbow joint of contralateral forelimb and another pair of electrodes was placed at the projection of the sural nerve at the back of the knee of the contralateral hindlimb. Nerves were stimulated with electrical rectangular pulses at 0.5-1 Hz, 1.0 ms with a current of 12-15 mA. All SSEPs were successfully registered in the window of 100 ms. An average of 150-200 responses was recorded at each stimulation protocol. Histological analysis At phase 4 (7 week after UCBC transplantation or 60 days after SCI) control and experimental animals were anesthetized with Zoletil®100 and intracardiacally perfused with 4% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS, pH 7.4). Spinal cord segments 5 cm rostral and 5 cm caudal from the contusion site were removed and postfixed in 4% paraformaldehyde at 4°C overnight. Semi-thin sections. For morphologic analysis 5-mm sections of the fixed spinal cord, 5 mm distal from the contusion site, were embedded in EMbed 812 (Electron Microscopy Sciences). The cross semi-thin sections were stained with methylene blue dye and studied by light microscopy. Changes in white mater were evaluated in 0.1mm2 area in the later part of lateral funiculus. Immunofluorescence. Spinal cord samples 10 mm caudal from injury were cryoprotected in 30% sucrose, frozen in liquid nitrogen, and embedded in tissue freezing medium. Frozen freefloating (20 μm) coronal serial sections of spinal cords were prepared using the cryostat Microm HM 560 (Thermo Scientific, Waltham, MA, USA). After blocking of non-specific binding sites using normal donkey serum, the primary antibodies (Abs) were applied overnight at 4°C. The subsequent incubation of the sections with proper secondary Abs for 2 h at room temperature was followed with cell nuclei staining with Propidium Iodide solution (PI, 5 μg/ml in PBS, Sigma). The list of primary and secondary Abs used in this study is presented in Table 1. After incubation with secondary Abs, sections were picked up on SuperFrost®Plus glass slides, mounted in anti-quenching medium and evaluated with a laser scanning microscope LSM 510 META (Carl Zeiss, Germany). Expression of HSP27 and HSP70 were blindly quantified in the ventral horns (VH), main corticospinal tract (CST), ventral funiculi (VF), area around the central canal (CC), and dorsal root entry zone (DREZ) in merged images from 10 adjacent optical slices (512 x 512 pixel resolution, observed area 0.05 mm2 ; acquisition distance, 0.5 µm). The meanintensity of labeling in the images was analyzed using the software Zen 2012 (Carl Zeiss). All sections were imaged in the z-plane by using identical confocal settings (laser intensity, gain, offset). For general morphology 5 μm slide mounted sections were stained with haematoxylin and eosin. Quantitative procedure for morphological analysis: Digitized images of semi-thin sections and frozen cross sections of spinal cord were obtained from one control animal with no therapy and one animal with treatment and were analyzed using the ImageJ software. The
total number of the preserved myelinated fibers in the ventral root was determined and presented as absolute meanings. Total frozen cross-sections, 25 micron thickness sections were performed at the 75 micron distance, totally 10 sections were collected along the 1 mm of spinal cord. The volume of the cavities was measured and converted into the percent data. Statistical analysis Student’s t-test was used in the statistical analysis with the values expressed as mean ±S.E.M. pvalues < 0.05 was considered statistically significant. Data were analyzed using Origin 7.0 SR0 (OriginLab, Northampton, MA, USA) software.
Results Behavioral and electrophysiological evaluation Before surgery the intact animals were active and mobile, with ability to stand, walk and run with an adequate balance and using all four limbs. No changes in sensation were diagnosed at this time point. During sensitivity tests, mini pigs showed a clear facial response, as well as a motor and vocal reaction without significant asymmetry. The range of active and passive movements of all limbs was normal. In SSEP test the amplitude of the first negative peak (N1) in microvolts (mkV) was measured from the baseline to the top of the peak, and the latency of N1 (in ms) was measured from the beginning of electrical stimulation to the first deviation from the baseline (Fig. 1A and B). When SSEPs were recorded in healthy animals before surgery, welldefined SSEP peaks were observed during stimulation of the nerves of both forelimbs and hind limbs. In healthy mini pig the value of the amplitude of the first negative SSEP peak during stimulation of the median nerve was 1.85 ± 0.22 mkV, for the sural nerve — 1.38 ± 0.02 mkV, and with the latency — 7.37 ± 0,24 ms and 11.48 ± 0.05 ms, for median and sural nerve respectively (Fig. 1A and B). No significant asymmetry between right and left side was found. Observed shorter latency of N1 response in forelimbs (Fig. 1A) over hind limbs (Fig. 1B) could be associated with the different length of conductive pathways. After surgery one day before UCBC transplantation, both animals presented significantly reduced locomotor activity and were unable to stand and walk using their hind limbs. Pain sensation assessment performed using skin pinch test on proximal and distal part of the hind limb revealed symmetrical loss of pain sensitivity with no vocal responses. Neurological examination revealed central type of paralysis of the hind limbs, loss of any active movement, increase tone in the extensor muscles of the hind limbs, symmetrical loss of pain sensitivity, and urinal and fecal incontinence. Bladder emptying was performed to all animals using Crede maneuver twice a day. No voluntary voiding was observed during the entire experiment. 3 weeks after UCBC transplantation in both control and experimental groups SSEPs assessment showed significantly reduced responses to the stimulation of the median nerve (1.38 ± 0.02 mkV) by 25% (p<0.05) and complete absence of SSEPs to the stimulation of the sural nerve (Fig. 1B). At five weeks after UCBC transplantation we did not observed any significant changes in neurophysiological assessment in control (SCI) and experimental (SCI with UCBC transplantation) mini pigs compare to three weeks assessment. At seven weeks after UCBC transplantation, we observed improvement in both behavioral and electrophysiological parameters in animal with UCBC transplantation, while control animal demonstrated no positive signs. While control untreated animal was using only the front legs, dragging the rear ones to move along the podium, the UCBC-treated mini pig showed successful attempts to stand and walk using hind limbs (Fig. 2). No movements in hip, knee and ankle joints were observed in control animal, wherein in the UCBC-treated mini pig the movements in these joints were confirmed by the changes in the angles value (Fig. 2). Functional recovery at seven weeks after UCBC transplantation was also correlated with restoration of pain sensitivity and improvement in electrophysiological evaluation. On SSEPs assessment at seven
weeks during stimulation of the sural nerve in UCBC-treated mini pig we observed well-defined N1 response (2.25 ± 0.02 mkV) (Fig. 1B). Locomotor function determined by PTIBS (Fleiss' kappa =0.94) corresponded to both behavioral and electrophysiological results in untreated and UCBC-treated mini pig (Fig. 1C). Histological evaluation Histological investigation revealed severe damage in gray and white mutter of spinal cord caused by contusion trauma. The analysis of light microscopy including immunofluorescent study revealed the differences in treated and control animals. Morphological analysis. Investigation of total cross sections of spinal cords 10 mm caudal from the epicenter showed diffuse intra and extracellular changes, such as cellularity reduction, presence of isolated hyaline balls, extension of perinuclear and pericellular spaces, tissue fragmentation with different in size cavities (Fig. 3). In the same time remarkable preservation of tissue in the gray and white mater was demonstrated in the treated animal. In the gray mater of the UCBC-treated mini pig a smaller volume of cystic cavities (41.49±1.01% vs. 71.38±2.34%) and superior tissue sparing (58.51±1.06% vs. 28.62±2.34%) were identified in compare with saline treated animal. Moreover in the white mater of the treated mini pig the number of clearly defined myelinated fibers were higher in comparison with the control animal (1119.50±4.32 vs. 767.33±2.80) (Fig. 4). Immunofluorescence. HNA: Staining of the spinal cord sections with Abs against human nuclei antigen (HNA) in order to observe the transplanted UCBC did not reveal any HNA-positive cells due to rejection of the UCBC by the host immunocompetent cells or limited UCBC survivability. In our previous study gene modified UCBC were detected in ALS mice spinal cord 17 weeks after intravenous administration [13]. We did not find UCBC in mini-pig spinal cord 8 weeks after intrathecal injection and several reasons may explain this, i.e. open surgery wound and inflammation may activate rejection of the grafted UCBC by the time of morphological investigation. However, based on our results obtained on rat model of SCI, HNA-positive cells were found 4 weeks after intrathecal injection of gene-modified UCBC overexpressing VEGF, GDNF and NCAM (unpublished observation). These findings suggest that following administration, the UCBC provide the recombinant therapeutic molecules and have time to produce VEGF and GDNF with neuroprotective effect and may increase survivability of the affected neurons. HSP27: In three areas of gray matter (VH — ventral horn, DREZ — dorsal root entering zone, and CC — central canal) immunoexpression of HSP27 was higher in the UCBC-treated mini pig. In the VH it was 3.15±0.40 vs. 5.28±1.02 (p<0.05), in the DREZ — 12.63±0.81 vs. 19.34±1.63 (p<0.05) and in the CC — 5.13±0.74 vs. 10.30±1.05 (p<0.05) (Fig. 5). The difference in the mean-intensity of HSP27 in white matter areas (VF — ventral funiculus and CST — corticospinal tract) between the experimental and control animals was insignificant. In the VF area it was 3.15±0.40 vs. 5.28±1.02 and in the CST — 5.29±0.18 vs. 7.31±0.81. HSP70: Compared with the control group, the expression of HSP70 was higher in the UCBC treated mini pig in DREZ (11.78±0.97 vs. 22.11±0.98, p<0.05), in VF (8.14±0.77 vs. 21.82±0.65, p<0.05) and particularly in CST (1.94±0.30 vs. 6.84±0.79, p<0.05) (Fig. 6). In contrast to the mean-intensity expression of HSP27, HSP70 expression was significantly higher in assessed areas of white matter in the UCBC-treated mini pig than in the control animal. Thus, the mean-intensity the number of HSP70 positive cells in the VF and CST in the experimental animal exceeded the number of similar cells in the control mini pig by more than 2- and 3-fold, respectively.
Discussion Searching for new SCI treatment strategies is the one of the most challenging directions in neuroscience and medicine. Currently available therapies are not significantly improving the quality of patients’ life neither prolongs its duration. The results of many studies designed to overcome the consequences of neurodegeneration in patients with SCI are extremely unsatisfactory and require development of new therapeutic protocols. Further improvement of techniques designed for neuroprotection, maintenance of neural fibers growth, and recovery of neural connections, become more dependent from the modern neuroengineering [16, 17, 18, 19, 20] and molecular genetic approaches [9, 10, 11, 21]. The main goal of the present study was to develop a novel therapeutic protocol for recovery of motor function after spinal cord injury and evaluate methodological aspects of implementation of this therapy in a large animal model of mini-pig. The spinal cord contusion model in mini pigs was chosen to test exclusive genetically engendered umbilical cord blood cells (UCBC) for stimulation of spinal cord neuroregeneration. In this pilot study for the first time we applied UCBC-based delivery of three therapeutic genes in mini-pig spinal cord injured model. We investigated the feasibility of intrathecal delivery of the gene modified UCBC on functional recovery, evaluated with behavioural and electrophysiological assessment, and performed histological post-mortem analysis (morphology and heat shock proteins immunoexpression) of spinal cord 60 days after trauma. Over the last years two common neurotrophins (neurotrophin-3 and brain-derived neurotrophic factor) and overexpression of neurotrophins and their receptors have been broadly used to stimulate neuroregeneration [8]. Although various stem and progenitor cells were suggested for transplantation into the nervous tissue as a carrier of therapeutic genes, UCBC were successfully used for the treatment of non-hematopoietic disorders, such as neurodegenerative and cardiovascular diseases. We chose UCBC for gene delivery in this study due to its availability for both allogeneic and autologous transplantation in humans, low immunogenicity, and simplicity in preparation and storage. Moreover, the lack of legal, ethical and religious restrictions for transplantation of umbilical cord blood cells is also an important reason for choosing these cells [4,22]. In addition, mononuclear cord blood has different stem cells, which can promote specialized cells for various tissues and/or can be a source for many growth and trophic factors [23,24,25]. Previously we utilized the gene-cell constriction based on UCBS transduced with three Ad-5 encoding VEGF, GDNF and NCAM for delivery of viral vectors carrying therapeutic genes into spinal cord of ALS mice [13]. In this construct the NCAM helps UCBC to survive and migrate to the sites of degeneration for production of VEGF and GDNF, thus tandem delivery of multiple genes is aimed to achieve different effects on neuroregeneration. It is expected that recombinant adhesion molecule NCAM can provide the targeted migration and survival in the neural tissue of a recipient of the transplanted UCBC, that will in turn produce the therapeutic molecules (VEGF and GDNF) in the area of trauma. VEGF and GDNF, the molecules with well-known neuroprotective mechanism preventing the entry of cells into apoptosis. Furthermore, VEGF plays an important part in restoring the microcirculation in the ischemic area after a neurotrauma [26]. Thus, NCAM supplied UCBC may provide targeted delivery of therapeutic genes to trauma area and act as a bioreactor for local overproduction of the growth factors. The impact UCBC-mediated gene therapy is addressed primary to avoid the post-traumatic neuronal death and in this study we have demonstrated for the first time the effect of proposed gene-cell construct therapy on recovery of mini pigs after SCI. To overcome a common problem of UCBC delivery across the brain-blood barrier, we used intrathecal method delivery, which was already tested in clinical trials for stem cell delivery [27]. Histological, electrophysiological, and clinical evaluation in this study demonstrated significant improvement in animal treated with genetically engineered UCBCs. Although presented results have been collected from two experimental animals, the motor recovery and improvement in SSEP in the treated mini pig provide methodological basis for future studies to support the development of
new gene-cell therapy for SCI. Future behavioral, electrophysiological, and more detailed histological investigation should be performed, but available results can be considered as an evidence of feasibility and potential efficacy of proposed therapy.One of the main mechanisms of gene-cell therapy is the induction of heat shock proteins (HSPs) expression. HSPs supposed to prevent the aggregation and denaturation of many proteins and appear to be a potent protective factor for neuronal cells. Endogenous protective molecules, such as HSP27 and HSP70, acting as molecular chaperones, have anti-apoptotic activity for neuronal cells of the CNS [28]. The present results suggest that UCBC-mediated gene delivery of Ad5-VEGF165, Ad5-GDNF and Ad5-NCAM1 increase the expression of HSP27 and HSP70 in injured spinal cord. This approach together with electrophysiological assessment on large animal models [29] represents a new experimental and therapeutic strategy for SCI. In summary, the intrathecal injection of the gene-cell constraction was for the first time used for SCI treatment in mini-pig SCI model. The efficacy of the treatment obtained in this pilot study provides confidence in future development of the method of post injury spinal cord repair. Although the more detailed behavioral, electrophysiological, and histological investigation should be performed, current results can be considered as an evidence of feasibility and potential efficacy of proposed therapy on large animal model. Data on the functional recovery in mini-pig SCI model after cell-mediated gene delivery to the spinal cord indicates that suggested therapy could be potentially implemented in patients with SCI. Acknowledgments. This study was supported by the grant of Russian Science Foundation No. 16-15-00010. We would like to thank Izmailov A.A., Kitaeva E.A., Bashirova E.Sh, and Boychuk S.V. for their assistance in some experiments.
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[16] Yu. Gerasimenko, V. Avelev, O. Nikitin, I. Lavrov. Initiation of locomotor activity in spinalized cats by epidural stimulation of the spinal cord, Ross Fiziol Zh Im I M Sechenova, 87 (2001) 1161-1170 [17] I Lavrov, PE Musienko, VA Selionov, RR Roy, VR Edgerton, YR Gerasimenko, Activation of spinal locomotor circuits in the decerebrated cat by spinal epidural and/or intraspinal electrical stimulation, Brain Research 1600 (2015) 84-92 [18] P Gad, IA Lavrov, P Shah, H Zhong, RR Roy, VR Edgerton, Y Gerasimenko, Neuromodulation of motor-evoked potentials during stepping in spinal rats, Journal of Neurophysiology 110(6) (2013) 1311-22 [19] M Nandra, I Lavrov, VR Edgerton, YC Tai, A parylene-based microelectrode array implant for spinal cord stimulation in rats, Proceedings of the IEEE Eng Med Biol Soc 2011 (2011) 1007-1010. [20] P Gad, J Woodbridge, I Lavrov, H Zhong, RR Roy, M Sarrafzadeh, VR Edgerton, Forelimb EMG-based trigger to control an electronic spinal bridge to enable hindlimb stepping after a complete spinal cord lesion in rats, Journal of Neuroengineering and Rehabilitation (2012) 9:38 [21] T. Federici, J. Riley, J. Park, M. Bain, N. Boulis, Preclinical Safety validation of a stabilized Viral vector direct Injection approach to the cervical spinal cord, Clin Transl Sci. 2(2) (2009) 165–167. [22] Y.H. Lee, Regenerative Potential of Intravenous Infusion with Mononuclear Cells in Cord Blood and G-CSF-Mobilized Peripheral Blood. J Stem Cell Res Ther 4 (224) (2014) http://dx.doi.org/10.4172/2157-7633.1000224. [23] C.G. Fan, Q.J. Zhang, F.W. Tang, Z.B. Han, G.S. Wang, Z.C. Han, Human umbilical cord blood cells express neurotrophic factors, Neurosci Lett. 380 (3) (2005) 322–325. [24] D.T. Harris, I. Rogers, Umbilical cord blood: a unique source of pluripotent stem cells for regenerative medicine, Curr Stem Cell Res Ther. 2 (4) (2007) 301–309. [25] S. Neuhoff, J. Moers, M. Rieks, T. Grunwald, A. Jensen, R. Dermietzel, C. Meier, Proliferation, differentiation, and cytokine secretion of human umbilical cord blood-derived mononuclear cells in vitro, Exp Hematol. 35 (7) (2007) 1119–1131. [26] S.A. Figley, Y. Liu, S.K. Karadimas, K. Satkunendrarajah, P. Fettes, S.K. Spratt, G. Lee, D. Ando, R. Surosky, M. Giedlin, M.G. Fehlings, Delayed administration of a bio-engineered zincfinger VEGF-A gene therapy is neuroprotective and attenuates allodynia following traumatic spinal cord injury, PLoS One 9 (5) (2014), http://dx.doi.org/10.1371/journal.pone.0096137. [27] M.A. Sahraian, M.M. Bonab, S.A. Karvigh, S. Yazdanbakhsh, B. Nikbin, J. Lotfi, Intrathecal Mesenchymal Stem Cell Therapy in Multiple Sclerosis: A Follow-Up Study for Five Years After Injection Arch, Neurosci. 1 (2) (2014) 71–75. [28] T.B. Franklin, A.M. Krueger-Naug, D.B. Clarke, A.P. Arrigo, R.W. Currie, The role of heat shock proteins Hsp70 and Hsp27 in cellular protection of the central nervous system, Int J Hyperthermia. 21 (5) (2005) 379–392. [29] F.D. Benavides, A.J. Santamaria, D.L. Guada, N. Bodoukhin, J. Solano, J. Guest, Characterization of motor and somatosensory evoked potentials in the Yucatan micropig using transcranial and epidural stimulation, J Neurotrauma. (2016) Epub ahead of print.
Figure legends and Tables Fig. 1. Upper panel — SSEP of animals during stimulation of the median (A) and the sural (B) nerves, where the solid line — healthy animal; the dashed line — UCBC-treated animal three weeks after surgery; the discontinuous line — UCBC-treated animal seven weeks after surgery; N1 – the first negative peak; Scale — 5ms/div. Lower panel (C) — PTIBS (Porcine Thoracic Injury Behavioral Scale) scores for the control (no treatment) animals and for the UCBC-treated animal.
Fig. 2. Evaluation of locomotor function in mini pig after contusion during the walking test in the 3 meters length podium. In the upper panel control animal with saline administration drags the body along the podium using only front legs. In the down panel the animal treated with gene modified UCBCs is using the rear legs during the walking test. Тhe hip, knee and ankle joint angles and changes in their values are shown.
Fig. 3. Mini pig lumbar spinal cord 10 mm caudal to the lesion epicenter at Day 60 post injury. Frozen 10 μm cross sections stained with hematoxylin and eosin. Left panel – control animal with saline administration. Right panel – experimental animal treated with gene modified UCBCs. Less developed cavities and more preserved tissue is seen in the spinal cord of UCBCtreated mini pig.
Fig. 4. White matter (lateral funiculus) 5 mm caudal to the lesion epicenter at Day 60 post injury. Control — animal with saline administration; Treatment — UCBC-treated animal. Semithin sections stained with toluidine blue.
Fig. 5. Immunoexpression of heat shock protein 27 (HSP27) in gray matter (VH, ventral horn, A–B; DREZ, dorsal root entry zone, C–D; CC, central canal, E-F) of a control animal (A, C, E) and an UCBC-treated animal (B, D, F) at 60 days after SCI. Sections through lumbar spinal cord show immunostaining for HSP27 (red). Nuclei are stained with Hoechst 33258 dye (blue). Scale bar, 20 µm.
Fig. 6. Immunoexpression of heat shock protein 70 (HSP70) in spinal cord (VH, ventral horn, A–B; DREZ, dorsal root entry zone, C–D; VF, ventral funiculus, E-F) of a control animal (A, C, E) and an UCBC-treated animal at 60 days after SCI (B, D, F). Sections through lumbar spinal cord show immunostaining for HSP70 (red). Nuclei are stained with Hoechst 33258 dye (blue). Scale bar, 20 µm.
Table 1. Primary and secondary antibodies used in immunofluoresent staining Antibody Host Dilution Source Hsp27 Rabbit 1:100 Sigma Hsp70 Rabbit 1:100 Sigma Human nuclei antigen (HNA) Mouse 1:150 Millipore Anti-rabbit Alexa 647 Donkey 1:200 Invitrogen Anti-mouse Alexa 647 Donkey 1:150 Invitrogen
S0304-3940(17)30140-4 http://dx.doi.org/doi:10.1016/j.neulet.2017.02.034 NSL 32646
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
22-6-2016 24-11-2016 12-2-2017
Please cite this article as: Islamov Rustem Robertovich, Sokolov Mikhail Evgenyevich, Bashirov Farid Vagizovich, Fadeev Filip Olegovich, Shmarov Maxim Michaylovich, Naroditskiy Boris Savelyevich, Povysheva Tatyana Vyacheslavovna, Shaymardanova Gulnara Ferdinantovna, Yakupov Radik Albertovich, Chelyshev Yuri Aleksandrovich, Lavrov Igor Aleksandrovich, A pilot study of cellmediated gene therapy for spinal cord injury in mini pigs, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2017.02.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A pilot study of cell-mediated gene therapy for spinal cord injury in mini pigs
Islamov Rustem Robertovich1*, Sokolov Mikhail Evgenyevich1,2, Bashirov Farid Vagizovich1, Fadeev Filip Olegovich1,2, Shmarov Maxim Michaylovich4, Naroditskiy Boris Savelyevich4, Povysheva Tatyana Vyacheslavovna1, Shaymardanova Gulnara Ferdinantovna1, Yakupov Radik Albertovich3, Chelyshev Yuri Aleksandrovich1,2, Lavrov Igor Aleksandrovich2,5*
1
Kazan State Medical University, Kazan, Russia
2
Kazan Federal University, Kazan, Russia
3
Kazan State Medical Academy, Kazan, Russia
4
Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia
5
Departments of Neurologic Surgery and Biomedical Engineering, Mayo Clinic, Rochester, MN,
US *
corresponding authors
*Correspondence (Igor Lavrov, MD, PhD): Address: Department of Neurologic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, US Tel: +(1) 507-538-7400 E-mail: [email protected]
Highlights
Intrathecal injection of the gene-cell constraction (UCBC-based delivery of three therapeutic genes) was used for the first time for SCI treatment in large animal mini-pig SCI model. Results provide evidence of feasibility and potential efficacy of proposed UCBC-based delivery of three therapeutic genes therapy. Recovery of the motor functions, somatosensory evoked potentials, and histological findings in control and treated animals support the positive effect of the gene-cell constriction for recovery after spinal cord injury in large animal model.
Abstract Currently, in clinical practice there is no efficient way to overcome the sequences of neurodegeneration after spinal cord traumatic injury. Using a new experimental model of spinal cord contusion injury on miniature pigs, we proposed to deliver therapeutic genes encoding vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM) to the damaged area, using umbilical cord blood mononuclear cells (UCBC). In this study, genetically engineered UCBC (2×106 cells in 200 µl of saline) were injected intrathecally to mini-pigs 10 days after SCI. Control and experimental mini pigs were observed for 60 days after surgery. Histological, electrophysiological, and clinical evaluation demonstrated significant improvement in animal treated with genetically engineered UCBCs. Difference in recovery of the somatosensory evoked potentials and in histological findings in control and treated animals support the positive effect of the gene-cell constriction for recovery after spinal cord injury. Results of this study suggest that transplantation of UCBCs simultaneously transduced with three recombinant adenoviruses Ad5-VEGF, Ad5-GDNF and Ad5-NCAM represent a novel potentially successful approach for treatment of spinal cord injury.
KEYWORDS: Cell-mediated gene therapy, adenoviral vector, Spinal Cord Injury, Mini pigs, Human umbilical cord blood mononuclear cell, Vascular endothelial growth factor (VEGF), Glial cell-derived neurotrophic factor (GDNF), Neural cell adhesion molecule (NCAM).
Introduction Spinal cord injury (SCI) is classified as a neurotrauma, although it is followed by disturbance in blood supply, neurodegenerative and other pathological consequences [1]. Regardless numerous animal studies and clinical trials, to this time there is no successful clinical protocol for SCI treatment. To enhance the efficacy of SCI therapy along with experiments on rodents and clinical trials, experiments on large animals models appear to be highly important. Most of the previous studies on neuroregeneration were performed on rodents and reported positive results require further investigations on large animal models similar in anatomy and physiology to human [2,3,4]. Between others available experimental models mini pigs are seem to be one of the optimal because of anatomic, physiologic and ethical reasons [2]. The importance of performing experiments on swine is dictated by several reasons, such as (1) anatomical considerations, thus, the length of neuritis in human is more then 100 times longer compare to rodent, (2) sharing with
human common physiological characteristics, (3) pharmaceutical reasons, such the dosage and the way of drug delivery is close to human. These details are vary from model to model and particularly between the small and large animals and the positive results obtained on rodents may not be directly applicable on patients [5,6]. Current progress in gene therapy for spinal cord injury aimed to protect neurons and stimulate the growth of neuritis [7]. Both the direct gene therapy, as well as a cell-mediated gene therapy for spinal cord injury, is actively studied during the last years. The therapeutic genes and types of cells for genes delivery are diverse, although the best gene or gene-cell constriction for stimulation of neuroregeneration is still has to be identified [8]. Up to date a big variety of cell types and viral vectors are employed for cell-based gene delivery into CNS in rodents. Direct gene therapy or cell-based gene delivery following the SCI was successful in preservation of neurons and neuronal fibers and in improving locomotion in rats [9,10,11]. In our previous experiments, using the model of spinal cord injury in rats and transgenic G93A mice with amyotrophic lateral sclerosis (ALS), we successfully developed the gene-cell construction based on umbilical cord blood mononuclear cells (UCBC) and adenoviral vectors expressing neurotrophic factors (VEGF and GDNF) and neural cell adhesion molecule (NCAM) [12]. Adenoviral vector carrying GDNF for direct gene or UCBC-mediated GDNF gene therapy of spinal cord contusion in rat showed advantages of both methods. Co-expression in UCBC of recombinant VEGF and NCAM or GDNF and NCAM also showed symptomatic improvement, increased life span and sustained cell homing after transplantation into ALS mice. Our results suggest that the tandem overexpression of VEGF, GDNF (as a therapeutic molecules) and NCAM (as a molecule for survivability and addressed homing of UCBC) in genetically engineered UCBC can be useful in treatment of SCI in mini pigs. Based on our previous results with UCBC-mediated gene therapy on SCI rat model, we proposed a pilot experiment on mini pigs with genetically engineered UCBC. The goal of this study was to develop and evaluate methodological aspects of a new treatment strategy of UCBS transplantation combined with three therapeutic genes in a large animal model of the mini-pig. To address this goal, we investigated the therapeutic efficacy of UCBC transduced with VEGF, GDNF and NCAM after intrathecal transplantation in mini-pigs with spinal cord contusion.
Methods This study was conducted in compliance with local IACUC protocol (institutional animal protocol at Kazan State Medical University (KSMU), # 5, from May 27, 2014) and IRB protocol based on the license of 'Stem Cell Bank' of KSMU. All pregnant women were signed informed consent for using their umbilical cord blood and were thoroughly examined before the delivery for possible contraindications for umbilical cord blood cell donation. Umbilical cord blood was collected by trained personnel of the maternity hospital according to the instruction of Stem Cell Bank of KSMU. Preparation of genetically modified human umbilical cord blood mononuclear cells Adenoviral vectors generation. The plasmids and the recombinant human adenoviruses of serotype 5 Ad5-VEGF165, Ad5-GDNF, and Ad5-NCAM1 were obtained using VEGF165, GDNF, and NCAM1 genes, respectively. The nucleotide sequences encoding VEGF165 (Gene Bank NM_001171626.1), GDNF (Gene Bank NM_019139.1) and NCAM1 (Gene Bank NM_001076682.2) were obtained by chemical synthesis in “Evrogen” JSC. The AdEasy Adenoviral Vector System was used to construct Ad5-VEGF165, Ad5-GDNF and Ad5-NCAM1 according to the manufacturer’s instructions. rAds were grown in HEK-293 and purified by exclusion chromatography. The titers of Ad5-VEGF165, Ad5-GDNF and Ad5-NCAM1(2×109 PFU/ml, 4×109 PFU/ml and 1×109 pfu/ml, respectively) were determined by the plaque formation technique in the HEK-293 cell culture.
Cell preparation. Umbilical cord blood was obtained from healthy women during normal delivery. The blood was processed in accordance to the protocol of the legitimate and ethical standards generally accepted in the stem cell bank of the Kazan State Medical University. Mononuclear fraction of human UCB was isolated by standard technique of sedimentation on to a density barrier (Ficoll, 1.077g/ml) as described previously [13]. After purification UCBCs were cultivated in RPMI-1640 medium supplemented with 10% FBS and mixture of antibiotics penicillin and streptomycin (100 U/ml, 100 micrograms/ml) (Sigma, USA). UCBC were seeded in 10 cm culture dish and simultaneously transduced with three recombinant adenoviruses Ad5VEGF, Ad5-GDNF and Ad5-NCAM at MOI 10. Afterwards cells were incubated for 12-16 hours in a humid environment at a temperature of +37°C with 5% CO 2 content, washed with DPBS solution and prepared for transplantation to experimental group of animals. Animals and treatments In the study 6-month-old female Vietnamese Pot-Bellied miniature pig weighing 5 kg were employed. For two weeks before the surgery animals were kept solely in a housing area with 12 h light/dark cycles, controlled temperature (24-25°C) and water/food supply. All experimental procedures were performed in accordance with the Kazan State Medical University Animal Care and Use Committee guidelines. Contusion spinal cord injury model. At the day of surgery pre-medication with intramuscular injection of ketamine (5 mg/kg) and diazepam (0.5 mg/kg) was performed. Antibiotic cefazolin (60 mg) was given intramuscular before surgery. Anesthesia was induced with propofol and afterwards animals were endotracheally intubated and maintained on isoflurane (1.3%). Rectal temperature was evaluated continuously during surgery and electrophysiological tests and regulated with thermal blankets. The operative area was cleaned with povidone-iodine, dressed with sterile material. Intravenous infusion and postoperative drug administration was performed via the venous catheter. An urinary catheter was placed during the surgery and was maintained post-operatively. Contusion model with weight-drop device was used in our group over last years and was designed based on spinal cord contusion rodent model described previously [3, 14]. Briefly, for SCI a laminectomy was performed at the Th9–Th10 vertebral level and a custom modified weight-drop device ensured falling of the 50-g weight from the 50 cm height on the spinal cord causing the contusion injury [15]. Cell transplantation procedures. Ten days after surgery saline or UCBC were injected intrathecaly under ketamine (5 mg/kg) and diazepam (0.5 mg/kg) anesthesia at the L4–L5 vertebral level. A control animal received intrathecal injection of saline and an experimental mini pig received 2Ч106 UCBC in 200 µl of saline [12]. Behavioral assessment After animal acclimatization, animals were trained for one hour per day for total five days to walk with body weight support. Locomotor function was determined according to PTIBS (Porcine Thoracic Injury Behavior Scale) [2] and muscle tone in front and rear limbs were evaluated. PTIBS assessment was performed once a week during nine weeks period, when each animal was walking through a narrow 10m long corridor with transparent borders, while recorded on a camera. During the PTIBS assessment visually all steps were evaluated and the inter-rater agreement between 5 raters was quantified with Fleiss' kappa. Signs of pain sensitivity (skin pinch test) estimated by vocal response and muscle tone in forelimbs and hind limbs were evaluated. Somatosensory Evoked Potential (SSEP)
For SSEP assessment we used 2-channel digital miniature electromyograph «Neuro-EMGMicro» (Neurosoft, Russia). The baseline somatosensory evoked potentials (SSEP) were evaluated a week before surgery. After the surgery SSEP assessment was performed 4 times: one day before UCBC transplantation (Phase 1), 3 weeks (Phase 2), 5 weeks (Phase 3), and 7 weeks (Phase 4) post UCBC transplantation. All areas of recording were prepared with rubbing ethanol. Standard electrodes were used for stimulation of the n. suralis and n. medianus. An active recording electrode was placed in projection of the somatosensory cortex on contrаlateral to stimulation side and the reference electrode was placed at the point of intersection of the perpendicular line from the active electrode to the sagittal line of the head. Electrodes were coated with an electroconductive gel and fixed with adhesive tape to the scalp. The stimulating electrodes were placed at the projection of the median nerve at the elbow joint of contralateral forelimb and another pair of electrodes was placed at the projection of the sural nerve at the back of the knee of the contralateral hindlimb. Nerves were stimulated with electrical rectangular pulses at 0.5-1 Hz, 1.0 ms with a current of 12-15 mA. All SSEPs were successfully registered in the window of 100 ms. An average of 150-200 responses was recorded at each stimulation protocol. Histological analysis At phase 4 (7 week after UCBC transplantation or 60 days after SCI) control and experimental animals were anesthetized with Zoletil®100 and intracardiacally perfused with 4% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS, pH 7.4). Spinal cord segments 5 cm rostral and 5 cm caudal from the contusion site were removed and postfixed in 4% paraformaldehyde at 4°C overnight. Semi-thin sections. For morphologic analysis 5-mm sections of the fixed spinal cord, 5 mm distal from the contusion site, were embedded in EMbed 812 (Electron Microscopy Sciences). The cross semi-thin sections were stained with methylene blue dye and studied by light microscopy. Changes in white mater were evaluated in 0.1mm2 area in the later part of lateral funiculus. Immunofluorescence. Spinal cord samples 10 mm caudal from injury were cryoprotected in 30% sucrose, frozen in liquid nitrogen, and embedded in tissue freezing medium. Frozen freefloating (20 μm) coronal serial sections of spinal cords were prepared using the cryostat Microm HM 560 (Thermo Scientific, Waltham, MA, USA). After blocking of non-specific binding sites using normal donkey serum, the primary antibodies (Abs) were applied overnight at 4°C. The subsequent incubation of the sections with proper secondary Abs for 2 h at room temperature was followed with cell nuclei staining with Propidium Iodide solution (PI, 5 μg/ml in PBS, Sigma). The list of primary and secondary Abs used in this study is presented in Table 1. After incubation with secondary Abs, sections were picked up on SuperFrost®Plus glass slides, mounted in anti-quenching medium and evaluated with a laser scanning microscope LSM 510 META (Carl Zeiss, Germany). Expression of HSP27 and HSP70 were blindly quantified in the ventral horns (VH), main corticospinal tract (CST), ventral funiculi (VF), area around the central canal (CC), and dorsal root entry zone (DREZ) in merged images from 10 adjacent optical slices (512 x 512 pixel resolution, observed area 0.05 mm2 ; acquisition distance, 0.5 µm). The meanintensity of labeling in the images was analyzed using the software Zen 2012 (Carl Zeiss). All sections were imaged in the z-plane by using identical confocal settings (laser intensity, gain, offset). For general morphology 5 μm slide mounted sections were stained with haematoxylin and eosin. Quantitative procedure for morphological analysis: Digitized images of semi-thin sections and frozen cross sections of spinal cord were obtained from one control animal with no therapy and one animal with treatment and were analyzed using the ImageJ software. The
total number of the preserved myelinated fibers in the ventral root was determined and presented as absolute meanings. Total frozen cross-sections, 25 micron thickness sections were performed at the 75 micron distance, totally 10 sections were collected along the 1 mm of spinal cord. The volume of the cavities was measured and converted into the percent data. Statistical analysis Student’s t-test was used in the statistical analysis with the values expressed as mean ±S.E.M. pvalues < 0.05 was considered statistically significant. Data were analyzed using Origin 7.0 SR0 (OriginLab, Northampton, MA, USA) software.
Results Behavioral and electrophysiological evaluation Before surgery the intact animals were active and mobile, with ability to stand, walk and run with an adequate balance and using all four limbs. No changes in sensation were diagnosed at this time point. During sensitivity tests, mini pigs showed a clear facial response, as well as a motor and vocal reaction without significant asymmetry. The range of active and passive movements of all limbs was normal. In SSEP test the amplitude of the first negative peak (N1) in microvolts (mkV) was measured from the baseline to the top of the peak, and the latency of N1 (in ms) was measured from the beginning of electrical stimulation to the first deviation from the baseline (Fig. 1A and B). When SSEPs were recorded in healthy animals before surgery, welldefined SSEP peaks were observed during stimulation of the nerves of both forelimbs and hind limbs. In healthy mini pig the value of the amplitude of the first negative SSEP peak during stimulation of the median nerve was 1.85 ± 0.22 mkV, for the sural nerve — 1.38 ± 0.02 mkV, and with the latency — 7.37 ± 0,24 ms and 11.48 ± 0.05 ms, for median and sural nerve respectively (Fig. 1A and B). No significant asymmetry between right and left side was found. Observed shorter latency of N1 response in forelimbs (Fig. 1A) over hind limbs (Fig. 1B) could be associated with the different length of conductive pathways. After surgery one day before UCBC transplantation, both animals presented significantly reduced locomotor activity and were unable to stand and walk using their hind limbs. Pain sensation assessment performed using skin pinch test on proximal and distal part of the hind limb revealed symmetrical loss of pain sensitivity with no vocal responses. Neurological examination revealed central type of paralysis of the hind limbs, loss of any active movement, increase tone in the extensor muscles of the hind limbs, symmetrical loss of pain sensitivity, and urinal and fecal incontinence. Bladder emptying was performed to all animals using Crede maneuver twice a day. No voluntary voiding was observed during the entire experiment. 3 weeks after UCBC transplantation in both control and experimental groups SSEPs assessment showed significantly reduced responses to the stimulation of the median nerve (1.38 ± 0.02 mkV) by 25% (p<0.05) and complete absence of SSEPs to the stimulation of the sural nerve (Fig. 1B). At five weeks after UCBC transplantation we did not observed any significant changes in neurophysiological assessment in control (SCI) and experimental (SCI with UCBC transplantation) mini pigs compare to three weeks assessment. At seven weeks after UCBC transplantation, we observed improvement in both behavioral and electrophysiological parameters in animal with UCBC transplantation, while control animal demonstrated no positive signs. While control untreated animal was using only the front legs, dragging the rear ones to move along the podium, the UCBC-treated mini pig showed successful attempts to stand and walk using hind limbs (Fig. 2). No movements in hip, knee and ankle joints were observed in control animal, wherein in the UCBC-treated mini pig the movements in these joints were confirmed by the changes in the angles value (Fig. 2). Functional recovery at seven weeks after UCBC transplantation was also correlated with restoration of pain sensitivity and improvement in electrophysiological evaluation. On SSEPs assessment at seven
weeks during stimulation of the sural nerve in UCBC-treated mini pig we observed well-defined N1 response (2.25 ± 0.02 mkV) (Fig. 1B). Locomotor function determined by PTIBS (Fleiss' kappa =0.94) corresponded to both behavioral and electrophysiological results in untreated and UCBC-treated mini pig (Fig. 1C). Histological evaluation Histological investigation revealed severe damage in gray and white mutter of spinal cord caused by contusion trauma. The analysis of light microscopy including immunofluorescent study revealed the differences in treated and control animals. Morphological analysis. Investigation of total cross sections of spinal cords 10 mm caudal from the epicenter showed diffuse intra and extracellular changes, such as cellularity reduction, presence of isolated hyaline balls, extension of perinuclear and pericellular spaces, tissue fragmentation with different in size cavities (Fig. 3). In the same time remarkable preservation of tissue in the gray and white mater was demonstrated in the treated animal. In the gray mater of the UCBC-treated mini pig a smaller volume of cystic cavities (41.49±1.01% vs. 71.38±2.34%) and superior tissue sparing (58.51±1.06% vs. 28.62±2.34%) were identified in compare with saline treated animal. Moreover in the white mater of the treated mini pig the number of clearly defined myelinated fibers were higher in comparison with the control animal (1119.50±4.32 vs. 767.33±2.80) (Fig. 4). Immunofluorescence. HNA: Staining of the spinal cord sections with Abs against human nuclei antigen (HNA) in order to observe the transplanted UCBC did not reveal any HNA-positive cells due to rejection of the UCBC by the host immunocompetent cells or limited UCBC survivability. In our previous study gene modified UCBC were detected in ALS mice spinal cord 17 weeks after intravenous administration [13]. We did not find UCBC in mini-pig spinal cord 8 weeks after intrathecal injection and several reasons may explain this, i.e. open surgery wound and inflammation may activate rejection of the grafted UCBC by the time of morphological investigation. However, based on our results obtained on rat model of SCI, HNA-positive cells were found 4 weeks after intrathecal injection of gene-modified UCBC overexpressing VEGF, GDNF and NCAM (unpublished observation). These findings suggest that following administration, the UCBC provide the recombinant therapeutic molecules and have time to produce VEGF and GDNF with neuroprotective effect and may increase survivability of the affected neurons. HSP27: In three areas of gray matter (VH — ventral horn, DREZ — dorsal root entering zone, and CC — central canal) immunoexpression of HSP27 was higher in the UCBC-treated mini pig. In the VH it was 3.15±0.40 vs. 5.28±1.02 (p<0.05), in the DREZ — 12.63±0.81 vs. 19.34±1.63 (p<0.05) and in the CC — 5.13±0.74 vs. 10.30±1.05 (p<0.05) (Fig. 5). The difference in the mean-intensity of HSP27 in white matter areas (VF — ventral funiculus and CST — corticospinal tract) between the experimental and control animals was insignificant. In the VF area it was 3.15±0.40 vs. 5.28±1.02 and in the CST — 5.29±0.18 vs. 7.31±0.81. HSP70: Compared with the control group, the expression of HSP70 was higher in the UCBC treated mini pig in DREZ (11.78±0.97 vs. 22.11±0.98, p<0.05), in VF (8.14±0.77 vs. 21.82±0.65, p<0.05) and particularly in CST (1.94±0.30 vs. 6.84±0.79, p<0.05) (Fig. 6). In contrast to the mean-intensity expression of HSP27, HSP70 expression was significantly higher in assessed areas of white matter in the UCBC-treated mini pig than in the control animal. Thus, the mean-intensity the number of HSP70 positive cells in the VF and CST in the experimental animal exceeded the number of similar cells in the control mini pig by more than 2- and 3-fold, respectively.
Discussion Searching for new SCI treatment strategies is the one of the most challenging directions in neuroscience and medicine. Currently available therapies are not significantly improving the quality of patients’ life neither prolongs its duration. The results of many studies designed to overcome the consequences of neurodegeneration in patients with SCI are extremely unsatisfactory and require development of new therapeutic protocols. Further improvement of techniques designed for neuroprotection, maintenance of neural fibers growth, and recovery of neural connections, become more dependent from the modern neuroengineering [16, 17, 18, 19, 20] and molecular genetic approaches [9, 10, 11, 21]. The main goal of the present study was to develop a novel therapeutic protocol for recovery of motor function after spinal cord injury and evaluate methodological aspects of implementation of this therapy in a large animal model of mini-pig. The spinal cord contusion model in mini pigs was chosen to test exclusive genetically engendered umbilical cord blood cells (UCBC) for stimulation of spinal cord neuroregeneration. In this pilot study for the first time we applied UCBC-based delivery of three therapeutic genes in mini-pig spinal cord injured model. We investigated the feasibility of intrathecal delivery of the gene modified UCBC on functional recovery, evaluated with behavioural and electrophysiological assessment, and performed histological post-mortem analysis (morphology and heat shock proteins immunoexpression) of spinal cord 60 days after trauma. Over the last years two common neurotrophins (neurotrophin-3 and brain-derived neurotrophic factor) and overexpression of neurotrophins and their receptors have been broadly used to stimulate neuroregeneration [8]. Although various stem and progenitor cells were suggested for transplantation into the nervous tissue as a carrier of therapeutic genes, UCBC were successfully used for the treatment of non-hematopoietic disorders, such as neurodegenerative and cardiovascular diseases. We chose UCBC for gene delivery in this study due to its availability for both allogeneic and autologous transplantation in humans, low immunogenicity, and simplicity in preparation and storage. Moreover, the lack of legal, ethical and religious restrictions for transplantation of umbilical cord blood cells is also an important reason for choosing these cells [4,22]. In addition, mononuclear cord blood has different stem cells, which can promote specialized cells for various tissues and/or can be a source for many growth and trophic factors [23,24,25]. Previously we utilized the gene-cell constriction based on UCBS transduced with three Ad-5 encoding VEGF, GDNF and NCAM for delivery of viral vectors carrying therapeutic genes into spinal cord of ALS mice [13]. In this construct the NCAM helps UCBC to survive and migrate to the sites of degeneration for production of VEGF and GDNF, thus tandem delivery of multiple genes is aimed to achieve different effects on neuroregeneration. It is expected that recombinant adhesion molecule NCAM can provide the targeted migration and survival in the neural tissue of a recipient of the transplanted UCBC, that will in turn produce the therapeutic molecules (VEGF and GDNF) in the area of trauma. VEGF and GDNF, the molecules with well-known neuroprotective mechanism preventing the entry of cells into apoptosis. Furthermore, VEGF plays an important part in restoring the microcirculation in the ischemic area after a neurotrauma [26]. Thus, NCAM supplied UCBC may provide targeted delivery of therapeutic genes to trauma area and act as a bioreactor for local overproduction of the growth factors. The impact UCBC-mediated gene therapy is addressed primary to avoid the post-traumatic neuronal death and in this study we have demonstrated for the first time the effect of proposed gene-cell construct therapy on recovery of mini pigs after SCI. To overcome a common problem of UCBC delivery across the brain-blood barrier, we used intrathecal method delivery, which was already tested in clinical trials for stem cell delivery [27]. Histological, electrophysiological, and clinical evaluation in this study demonstrated significant improvement in animal treated with genetically engineered UCBCs. Although presented results have been collected from two experimental animals, the motor recovery and improvement in SSEP in the treated mini pig provide methodological basis for future studies to support the development of
new gene-cell therapy for SCI. Future behavioral, electrophysiological, and more detailed histological investigation should be performed, but available results can be considered as an evidence of feasibility and potential efficacy of proposed therapy.One of the main mechanisms of gene-cell therapy is the induction of heat shock proteins (HSPs) expression. HSPs supposed to prevent the aggregation and denaturation of many proteins and appear to be a potent protective factor for neuronal cells. Endogenous protective molecules, such as HSP27 and HSP70, acting as molecular chaperones, have anti-apoptotic activity for neuronal cells of the CNS [28]. The present results suggest that UCBC-mediated gene delivery of Ad5-VEGF165, Ad5-GDNF and Ad5-NCAM1 increase the expression of HSP27 and HSP70 in injured spinal cord. This approach together with electrophysiological assessment on large animal models [29] represents a new experimental and therapeutic strategy for SCI. In summary, the intrathecal injection of the gene-cell constraction was for the first time used for SCI treatment in mini-pig SCI model. The efficacy of the treatment obtained in this pilot study provides confidence in future development of the method of post injury spinal cord repair. Although the more detailed behavioral, electrophysiological, and histological investigation should be performed, current results can be considered as an evidence of feasibility and potential efficacy of proposed therapy on large animal model. Data on the functional recovery in mini-pig SCI model after cell-mediated gene delivery to the spinal cord indicates that suggested therapy could be potentially implemented in patients with SCI. Acknowledgments. This study was supported by the grant of Russian Science Foundation No. 16-15-00010. We would like to thank Izmailov A.A., Kitaeva E.A., Bashirova E.Sh, and Boychuk S.V. for their assistance in some experiments.
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Figure legends and Tables Fig. 1. Upper panel — SSEP of animals during stimulation of the median (A) and the sural (B) nerves, where the solid line — healthy animal; the dashed line — UCBC-treated animal three weeks after surgery; the discontinuous line — UCBC-treated animal seven weeks after surgery; N1 – the first negative peak; Scale — 5ms/div. Lower panel (C) — PTIBS (Porcine Thoracic Injury Behavioral Scale) scores for the control (no treatment) animals and for the UCBC-treated animal.
Fig. 2. Evaluation of locomotor function in mini pig after contusion during the walking test in the 3 meters length podium. In the upper panel control animal with saline administration drags the body along the podium using only front legs. In the down panel the animal treated with gene modified UCBCs is using the rear legs during the walking test. Тhe hip, knee and ankle joint angles and changes in their values are shown.
Fig. 3. Mini pig lumbar spinal cord 10 mm caudal to the lesion epicenter at Day 60 post injury. Frozen 10 μm cross sections stained with hematoxylin and eosin. Left panel – control animal with saline administration. Right panel – experimental animal treated with gene modified UCBCs. Less developed cavities and more preserved tissue is seen in the spinal cord of UCBCtreated mini pig.
Fig. 4. White matter (lateral funiculus) 5 mm caudal to the lesion epicenter at Day 60 post injury. Control — animal with saline administration; Treatment — UCBC-treated animal. Semithin sections stained with toluidine blue.
Fig. 5. Immunoexpression of heat shock protein 27 (HSP27) in gray matter (VH, ventral horn, A–B; DREZ, dorsal root entry zone, C–D; CC, central canal, E-F) of a control animal (A, C, E) and an UCBC-treated animal (B, D, F) at 60 days after SCI. Sections through lumbar spinal cord show immunostaining for HSP27 (red). Nuclei are stained with Hoechst 33258 dye (blue). Scale bar, 20 µm.
Fig. 6. Immunoexpression of heat shock protein 70 (HSP70) in spinal cord (VH, ventral horn, A–B; DREZ, dorsal root entry zone, C–D; VF, ventral funiculus, E-F) of a control animal (A, C, E) and an UCBC-treated animal at 60 days after SCI (B, D, F). Sections through lumbar spinal cord show immunostaining for HSP70 (red). Nuclei are stained with Hoechst 33258 dye (blue). Scale bar, 20 µm.
Table 1. Primary and secondary antibodies used in immunofluoresent staining Antibody Host Dilution Source Hsp27 Rabbit 1:100 Sigma Hsp70 Rabbit 1:100 Sigma Human nuclei antigen (HNA) Mouse 1:150 Millipore Anti-rabbit Alexa 647 Donkey 1:200 Invitrogen Anti-mouse Alexa 647 Donkey 1:150 Invitrogen