Edited by: Dimitrios I. Zeugolis, National University of Ireland Galway, Ireland
Reviewed by: Lisa Jane White, University of Nottingham, United Kingdom; Chiara Tonda-Turo, Politecnico di Torino, Italy
†These authors share first authorship
‡These authors share senior authorship
This article was submitted to Tissue Engineering and Regenerative Medicine, a section of the journal Frontiers in Bioengineering and Biotechnology
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Cervical spinal cord trauma represents more than half of the spinal cord injury (SCI) cases worldwide. Respiratory compromise, as well as severe limb motor deficits, are among the main consequences of cervical lesions. In the present work, a Gellan Gum (GG)-based hydrogel modified with GRGDS peptide, together with adipose tissue-derived stem/stromal cells (ASCs) and olfactory ensheathing cells (OECs), was used as a therapeutic strategy after a C2 hemisection SCI in rats. Hydrogel or cells alone, and a group without treatment, were also tested. Four weeks after injury, compound muscle action potentials (CMAPs) were performed to assess functional phrenic motor neuron (PhMN) innervation of the diaphragm; no differences were observed amongst groups, confirming that the PhMN pool located between C3 and C5 was not affected by the C2 injury or by the treatments. In the same line, the vast majority of diaphragmatic neuromuscular junctions remained intact. Five weeks post-injury, inspiratory bursting of the affected ipsilateral hemidiaphragm was evaluated through EMG recordings of dorsal, medial and ventral subregions of the muscle. All treatments significantly increased EMG amplitude at the ventral portion in comparison to untreated animals, but only the combinatorial group presented increased EMG amplitude at the medial portion of the hemidiaphragm. No differences were observed in forelimb motor function, neither in markers for axonal regrowth (neuronal tracers), astrogliosis (GFAP) and inflammatory cells (CD68). Moreover, using Von Frey testing of mechanical allodynia, it was possible to find a significant effect of the group combining hydrogel and cells on hypersensitivity; rats with a SCI displayed an increased response of the contralateral forelimb to a normally innocuous mechanical stimulus, but after treatment with the combinatorial therapy this behavior was reverted almost to the levels of uninjured controls. These results suggest that our therapeutic approach may have beneficial effects on both diaphragmatic recovery and sensory function.
Cervical spinal cord injuries (SCI) represent more than half of the SCI cases worldwide (
Among the existing works in traumatic cervical SCI, cellular transplantation strategies have been designed to address the following purposes: replacing or inducing plasticity of neurons involved in respiratory circuits (
Our group has developed a tissue engineering (TE) strategy for SCI repair, previously tested in a rat model of lumbar injuries (
Based on the abovementioned results, we determine whether the application of the same therapeutic strategy to a cervical hemisection injury could result in a beneficial outcome, modulating the local environment and possibly favoring neuronal preservation and/or inducing axon growth through the injury or in spared pathways. Toward this aim, following a C2 hemisection, ASCs and OECs encapsulated in the GG hydrogel were transplanted into the spinal cord lesion and functional and histological recovery was assessed.
Human ASCs were isolated according to
Olfactory ensheathing cells were isolated and cultured as previously described (
The synthesis of GG-GRGDS hydrogel was performed according to the protocols described by
GG-GRGDS lyophilized powder was sterilized by exposure to UV lights for 15 min, a method previously used without affecting the material properties (
As previously mentioned, all experimental procedures were approved either by the ICVS research committee and by the Thomas Jefferson University IACUC, being conducted in compliance with ARRIVE (Animal Research: Reporting of
Ten weeks old female Wistar-Han rats (forelimb motor studies, Charles River, France) or twelve weeks old Sprague-Dawley (functional diaphragm studies, Taconic, United States), housed in light and temperature-controlled rooms and fed with standard diet, were used in the
Experimental layout, with number of animals used per test, time points and rat breeds used in each set.
Staircase test | 8–10/group | 2 and 5 weeks | CMAPs | 5–8/group | 4 weeks | |
Grooming test | 8–9/group | 3 weeks | NMJ morphology | 3/group | 5 weeks | |
Von Frey test | 7–10/group | 4 weeks | EMGs | 6–8/group | 5 weeks | |
GFAP and CD68 | 3/group | 5 weeks | 5-HT sprouting | 4/group | 5 weeks |
Total number of animals used per test, for each experimental group.
Staircase test | 8 | 8 | 9 | 10 |
Grooming test | 8 | 8 | 8 | 9 |
Von Frey test | 7 | 8 | 7 | 10 |
GFAP and CD68 | 3 | 3 | 3 | 3 |
CMAPs | 6 | 8 | 7 | 5 |
NMJs morphology | 3 | 3 | 3 | 3 |
EMGs | 6 | 8 | 7 | 8 |
5-HT sprouting | 4 | 4 | 4 | 4 |
All animals were anesthetized by intraperitoneal injection of a mixture (1.5:1) of ketamine (100 mg/ml, Imalgene/Merial, France) and medetomidine hydrochloride (1 mg/ml, Domitor/Pfizer, United States). Once anesthetized, the dorsal surface of the skin was shaved and disinfected with a 70% ethanol solution and topical iodine (Dynarex, Orangeburg, New York). Using a sterile #11 surgical blade (Electron Microscopy Sciences, Hatfield, PA, United States), a three-cm midline incision was made on the dorsal surface of skin and muscle, starting from the caudal portion of the occipital bone. Retractors were then used to expose the dorsal surface of the C2 and C3 vertebrae. Using rongeurs (Fine Science Tools, Foster City, CA, United States), remaining tissue was removed from the vertebrae and a laminectomy was performed to expose the spinal cord. The C2 and C3 dorsal roots were located, and a hemisection was performed at a location just caudal to the C2 root with a dissecting knife (Fine Science Tools, Foster City, CA, United States). To ensure a complete hemisection, a 30-gauge needle (BD Biosciences, San Jose, CA, United States) was passed through the injury several times. Within each set, animals were divided into four different groups according to the treatment/procedure: (1) animals subjected to SCI (hemisection) with no treatment (HS); (2) SCI animals treated with GG-GRGDS (GG-GRGDS); (3) SCI animals transplanted with cells (ASCs/OECs); (4) SCI animals treated with ASCs and OECs encapsulated in GG-GRGDS (GG-GRGDS + ASCs/OECs). The total animals per set and group is described in
Total number of animals used per group and in each experimental set.
HS | 8 | 6 | |
GG-GRGDS | 8 | 8 | |
ASCs/OECs | 9 | 7 | |
GG-GRGDS + ASCs/OECs | 10 | 8 | |
Total | 35 | 29 | 64 |
Following SCI surgery and treatment, post-operative care was performed as previously described (
Three weeks after injury, animals were anesthetized as described in cervical injuries and subjected to intra-brainstem injections of AAV2-mCherry, as described previously (
Four weeks post-surgery, rats were anesthetized with isoflurane (Piramal Healthcare, Bethlehem, PA, United States) at a concentration of 3.0–3.5% diluted in oxygen. Animals were placed supine and the region just below the rib cage was shaved and cleaned with 70% ethanol. Phrenic nerve conduction studies were performed with stimulation at the neck via near nerve needle electrodes placed along the phrenic nerve. A reference electrode was placed on the shaved surface of the right costal region. The phrenic nerve was stimulated with a single burst at 6 mV (amplitude) for a 0.5 ms duration. Each animal was stimulated between 10 and 20 times to ensure reproducibility, and recordings were averaged for analysis. Animals were daily followed for any signs of distress in response to this procedure. ADI Powerlab 8/30 stimulator and BioAMPamplifier (ADInstruments, Colorado Springs, CO, United States) were used for both stimulation and recording, and Scope 3.5.6 software (ADInstruments, Colorado Springs, CO, United States) was used for subsequent data analysis. An additional control animal without lesion (laminectomy only) was used as an example of a normal CMAP recording.
Five weeks post-surgery and immediately before sacrifice, animals were anesthetized with isoflurane at a concentration of 3.0–3.5% diluted in oxygen. All animals had fully recovered from CMAP recordings, with no signs of distress. Once deeply anesthetized, a laparotomy was performed to expose the right hemi-diaphragm. Bipolar electrodes spaced 3 mm apart were placed for recording in three separate sub-regions of the hemi-diaphragm: dorsal, medial, and ventral. Electrodes were always placed at the endplate band at all recording locations. Two recordings were averaged over a 2-min time frame for each animal, and peak amplitude, burst duration and frequency were taken. Using LabChart 7 software (AD Instruments, Colorado Springs, CO, United States), the EMG signal was amplified and filtered through a band-pass filter (50–3000 Hz). Following recordings, animals were immediately euthanized with a triple-dose of ketamine/xylazine/acepromazine and the spinal cord and diaphragm were collected after transcardial perfusion with 4% paraformaldehyde. As in CMAP recordings, an additional control animal without lesion (laminectomy only) was used as an example of a normal EMG recording.
The staircase (also called skilled paw reaching test) was performed with double staircase boxes (Campden Instruments, Lafayette, IN, United States), as previously described (
Three weeks after injury grooming behavior was evaluated for all rats. A soft gauze with fresh tap water was applied to the rats’ back head to induce grooming. Then the rats were placed in a glass cylinder and their behavior was filmed. After two complete cycles of grooming (starting by licking the paws and ending by cleaning behind the ears) the rats were placed back in their cages. Later, both forelimbs were scored using a scale varying from 0 (unable to touch the nose) up to 5 (reach the back of their ears), adapting a protocol from
The Von Frey test was performed at 4 weeks post-injury, as described in
where Xf = value (in log units) of the final von Frey filament; k = tabular value corresponding to pattern of positive and negative responses [X and 0 sequence; consult (
In both experimental sets, animals were sacrificed 5 weeks post-injury/treatment.
Four animals per group were used for NMJ analysis. Rats were euthanized with a mixture of ketamine/xylazine/acepromazine. Animals were placed supine; two incisions were made into the skin and underlying muscle starting from the xyphoid process and extending laterally along the rib cage to expose the right hemidiaphragm. The right hemi-diaphragm was excised using spring scissors (Fine Science Tools, Foster City, CA, United States), stretched flat and pinned down on silicon-coated 10 cm dishes, and washed with PBS (Gibco, Pittsburgh, PA, United States). Next, a 20-min fixation in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA, United States) was performed, followed by several washes in PBS. After washing, superficial fascia was carefully removed from the surface of the diaphragm with Dumont #5 Forceps (Fine Science Tools, Foster City, CA, United States).
Whole-mount immunohistochemistry was performed, as described previously (
We morphologically evaluated NMJ innervation by quantifying three phenotypes at individual synapses: intact NMJs, completely denervated NMJs, and partially denervated NMJs. We identified intact NMJs by complete overlap of the pre-synaptic axon labeling with the post-synaptic α-bungarotoxin labeling. We defined completely denervated NMJs by total absence of overlap between pre- and post-synaptic marker labeling. Partially denervated NMJs showed some overlap of pre- and post-synaptic labeling; however, this overlap was not complete as observed with intact NMJs. For each muscle/animal, we quantified 200–300 NMJs across the entire hemi-diaphragm, and we expressed the data for each phenotype as the percentage of total NMJs. Whole-mounted diaphragms were imaged on a FluoView FV1000 confocal microscope (Olympus, Center Valley, PA, United States). We conducted NMJ analysis on ipsilateral hemi-diaphragm because previous published work showed no denervation or sprouting in contralateral hemi-diaphragm after cervical injury (
Five weeks post-injury, a rough dissection of the spine and spinal cord was performed in PFA perfused animals, centered on the site of hemisection and the tissues were additionally fixed in 4% PFA overnight. A more detailed dissection of the spinal cord was then conducted and the tissues were carefully placed on a solution of saccharose at 30% (w/v). After 24 h, 2.5–3 cm length of spinal cord tissues, centered on the lesion, were involved in frozen section medium (Neg-50, Thermo Scientific, United States), frozen with liquid nitrogen and stored at −20°C. Later on, longitudinal cross sections of 20 μm thickness were performed using a Leica CM1900 cryostat.
Frozen longitudinal sections of cervical spinal cord were air-dried and washed three times (5 min each) with PBS. Samples were then incubated in blocking solution (5% normal goat serum and 0.4% Triton X-100 diluted in PBS) for 1 h at room temperature. Sections were incubated overnight at 4°C with primary antibodies in blocking solution. The following primary antibodies were used: polyclonal rabbit 5-HT antibody (Immunostar, Hudson, WI, United States), polyclonal rabbit anti-Glial Fibrillary Acidic Protein (GFAP, Dako, Denmark) and monoclonal mouse anti-CD68 (Merck Millipore, MA, United States). Sections were then washed with PBS (three washes, 5 min each) and incubated with the following secondary antibodies conjugated to Alexa fluorophores (Invitrogen) in blocking solution for 1 h at room temperature: AF488 goat anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove, PA, United States) for 5-HT, AF488 goat anti-rabbit (Invitrogen) for GFAP and AF594 goat anti-mouse (Invitrogen) for CD68. After washing with PBS (three washes, 5 min each), sections were coverslipped. For 5-HT staining, images were acquired with a Zeiss Imager M2 upright microscope, and MetaMorph software was used to quantify 5-HT immunostaining. For quantification of CD68+ reactive macrophages/monocytes, and GFAP+ cells, tissue sections were imaged on an Olympus IX81 inverted microscope. The total area of CD68 and GFAP expressing cells was quantified in 4–6 slices per animal.
All statistical analyses were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, United States). Differences among groups were assessed by one-way ANOVA test or by the two-way ANOVA test followed by the Bonferroni
Lesions at the C2 spinal cord level are not expected to directly affect the PhMN pool located between C3 and C5. Therefore, in order to confirm the functional innervation of the ipsilateral hemi-diaphragm, CMAPs were recorded after supramaximal stimulation of the phrenic nerve. No significant alterations were observed among groups regarding CMAP amplitudes (
Cervical hemisection injury at C2 level
NMJ morphology in the ipsilateral hemi-diaphragm. No significant alterations were seen among groups and most of the NMJs were intact. Nicotinic acetylcholine receptors stained with α-bungarotoxin (in blue,
Five weeks after injury, diaphragmatic function was assessed through EMG analysis. After recordings at three different subregions of the hemi-diaphragm ipsilateral to injury, it was observed a clear reduction in EMG amplitudes following SCI (
EMG recovery in the ipsilateral hemi-diaphragm, 5 weeks after C2 cervical hemisection injury.
Serotonergic descending input plays an important role in the excitability of the spinal cord motor neurons, including PhMNs. Therefore, 5-HT axon sprouting was assessed at the ipsilateral lesioned side of the spinal cord specifically within the PhMN pool. Interestingly, all treatments induced an increase in the number and length of 5-HT fibers caudal to the lesion site in the ipsilateral C3-C5 ventral horn (
Serotonergic axon sprouting caudal to the ipsilateral lesion site.
Motor impairment of the forelimbs is another significant consequence of cervical SCI. In the C2 hemisection, only the right forelimb was expected to be affected. In order to evaluate the recovery of motor function, two different motor behavior paradigms were performed. The first one was the staircase test, in which rats under food restriction are assessed for their fine motor skills, such as reaching and grasping abilities, to retrieve and eat a sugared pellet. At 2 and 5 weeks after injury, rats were subjected to the test, where during the first 5 days they could retrieve pellets from both right and left sides and in the last 2 days, pellets were only available on the left or on the right side (forced choice,
Staircase test for the right paw, performed at
The second motor paradigm used was the grooming test. Grooming is a natural behavior in rodents that can be used to assess forelimb movement capacity. Using a pre-defined scale, where 1 means limited movements, while 5 represents a normal movement with complete grooming cycles, rats were evaluated at 3 weeks post-injury (
Grooming test performed at 3 weeks after injury.
Overall, these motor paradigms reinforce the idea that the injury was correctly performed, but treatments were unable to induce any motor recovery.
A common outcome following SCI is sensory dysfunction. With the objective of evaluating the response to mechanical stimuli, rats were subjected to the Von Frey test 4 weeks post-lesion. Following C2 cervical injury, it was observed a marked increase in sensitivity of the contralateral limbs (
Von Frey test performed at 4 weeks after injury. Forelimb sensory responses to Von Frey filaments were assessed and quantified. The combinatorial treatment led to a recovered sensory phenotype for the contralateral forelimb. Non-lesioned rats were used as a control. Data is presented as mean ± SEM (
Cervical SCI pre-clinical research is of great clinical relevance since lesions at this level account for more than half of SCI cases every year (
In our case, the combination of ASCs and OECs with the peptide-grafted GG hydrogel has led to relevant functional and histological improvements in other models of SCI (
While most clinical cases of SCI are contusive in nature, the C2 hemisection is a powerful model to investigate mechanisms of axon regeneration and sprouting as it creates a very precise and restricted injury, as well as complete ablation of defined axonal pathways. C2 hemisection interrupts the input of rVRG neurons from the brainstem, to PhMNs located around C3-C5 segments resulting in paralysis of the ipsilateral hemidiaphragm. In this sense, as a first experiment, the functionality and morphology of diaphragmatic innervation by PhMNs were analyzed (
Regarding limb motor function analysis, none of the therapeutic strategies employed impacted motor recovery. The motor behavior paradigms used address general forelimb movements (grooming test) but also skilled and fine detailed movements (staircase test). The specificity of the cervical neuronal circuitry might explain the different results observed between cervical and thoracic/lumbar studies. Forelimb motor function is highly dependent on supraspinal inputs, while thoracic and lumbar-derived movements rely significantly on local circuitries, such as central pattern generator (CPG) activity (
Interestingly, sensorial perception of the contralateral forelimb was also normalized following the combined treatment (
Cells from non-autologous sources were used in this study, both human ASCs and rat pup derived OECs. The use of autologous cells could have impacted differently the results observed, as it is expected that cell rejection rates are less significant. Still, we previously demonstrated that human ASCs can be integrated into the spinal cord tissue and survive for more than 8 weeks (
Cervical SCI are the frequent in humans, therefore, there is a need for more studies focusing on traumatic lesions affecting this spinal cord region. TE approaches such as the one presented here are promising, as they can address multiple targets, increasing the chances for functional recovery following an injury. In our work, the combination of GG-GRGDS hydrogel with ASCs/OECs led to an increased diaphragmatic activity together with a partial reestablishment of sensory function compromised by the injury. This is of the utmost relevance, as respiratory compromise and chronic pain are two of the main concerns of SCI patients. The specific mechanism(s) by which this therapeutic strategy exerts its effects may be related – at least in part – to an increase in specific serotonergic fiber sprouting in the ipsilateral caudal spinal cord. This strategy opens a window for improvement of a critical condition such as cervical SCI.
The datasets generated for this study are available on request to the corresponding author.
Human lipoaspirates obtained from consenting donors under an institutional review board approved protocol at LaCell LLC.
EG and BG contributed to the experimental design, data collection, analysis and interpretation, and drafting of the manuscript. RL, MG, TM-G, JM-M, MU, and MW contributed to the data collection, analysis, and interpretation. JG and NS provided the technical knowledge and materials for the experiments. NAS, AL, and AS interpreted the data, supervised the work, and revised the manuscript. All the authors approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
mCherry-labeled rVrg axons detected at the intact rostral spinal cord tissue, 5 weeks post-injury. Only a limited number of axons entered the lesion site, not growing through the lesion. No differences were observed amongst groups.
Quantification of the total area occupied by Gfap+