II. MATERIALS AND METHODS
12. Perfusion and tissue processing
Following intracardiac perfusion of the animals, spinal cords were carefully dissected, postfixed overnight in 4% paraformaldehyde, dehydrated overnight at 4°C in 30% sucrose, and frozen in isopentane. Two centimeter blocks of the thoracic region of the cords including injury epicenters were embedded in Tissue-Tek® OCT compound (Sakura Finetek, Torrence, CA.
USA) and cryosectioned for a tissue thickness of 20 µm.
13.
Histopatholigcal Analysis
Histopatholgy was analyzed after staining for hematoxylin and eosin (H&E) and solvent blue. Sections were imaged under a microscope (Carl-Zeiss USA) and digital photographs were taken and subsequently analyzed with Adobe® Photoshop® CS4 11.0.1 (Adobe, San Jose, CA. USA). Quantifications of lesion volume and white matter sparing as well as motor neuron quantification were performed on the three representative animals in each treatment group (whose behavior values most closely approximated the mean for that group). Lesion volume and white matter sparing were approximated by a method of pixel number comparisons between tissue and background (Fig. 2A
& B). Motor neuron quantification was performed separately for each spinal cord by counting the number of neurons with motor neuron morphology residing in the anterior horn of each side of the spinal cords (Fig. 2C).
A B C
Figure 2. Assessment of lesion volume (A), spared white matter (B), and motor neurons (C).
14.
Immunocytochemistry
Immunocytochemistry was performed on 20 µm mounted sections.
Primary antibodies for inflammatory markers were against glial fibrillary acidic protein (anti-GFAP rabbit; Millipore; 1:1000), CD11b (anti-CD11b mouse; AbD Serotec; 1:250), CD68 (anti-CD68 mouse; Chemicon; 1:250) and nitrotyrosine (anti-NT mouse; Santa Cruz; 1:250). ICC for endogenous stem cell activity was performed with primary antibodies against nestin (anti-nestin mouse; Santa Cruz; 1:200) and doublecortin (anti-DCX goat; Santa Cruz; 1:250).
Angiogenesis was evaluated with primary antibodies to laminin (anti-laminin rabbit; Sigma; 1:60) and CD31 (anti-CD31 goat; Santa Cruz; 1:400).
Neurotrophic activities in the transplants were evaluated with ICC primary antibodies against BDNF (anti-BDNF chicken; Promega; 1:250) and IL10 (anti-IL10 mouse; Santa Cruz; 1:250). Antibodies to evaluate donor cell fate included: collagen 1(anti-col1 rabbit), collagen 2 (anti-col2 mouse), and
alkaline phosphatase (ALP) (anti-ALP) (all Santa Cruz; 1:250) as well as for lipids with Oil Red O (Sigma). Co-staining for nuclei was performed with DAPI (Vectashield), for CD90 (anti-CD90 goat; Santa Cruz; 1:300) and HSP (anti-HSP 27 rabbit; Stressgen bioreagents; 1:250). Secondary antibodies included: donkey anti-rabbit FITC, donkey anti-mouse Texas Red, donkey anti-mouse FITC, Dylight 594 donkey anti-goat, donkey anti-chicken TR, donkey anti-mouse FITC and donkey anti-rabbit Texas Red (all Jackson Immunoresearch and 1:250). Primary antibodies were incubated at 4 degrees C overnight, followed by secondary antibody incubation at room temperature for 1 hr. Blocking was performed for 1 hr at room temperature immediately before primary antibody incubation. Blocking solution consisted of donkey serum with 5% bovine serum albumin. Imaging was performed either with a fluorescent microscope or confocal microscope (Carl-Zeiss USA). Semi-quantification of immunocytochemistry was performed by measuring signal intensity above a threshold level, and dividing these numbers of pixels but the total pixel count, to yield a percentage above threshold, or relative signal value that is reported in the results.
15.
Donor hMSC Survival
Immunocytochemisty of scaffold+hMSC spinal cords was performed for HSP and DAPI, and the number of surviving cells were reviewed with a 5X micrographs of 20 µm sections at consecutive millimeters on either side of the
section. The number of surviving human cells was estimated but dividing the spinal cord into 8 sectors and manually estimating the cell number in each sector. These numbers were summed to represent the total number of survival donor cells in the section. The same method was used with CD90 to confirm the trend of donor cell survival in a spatial relationship.
16.
Anterograde and Retrograde Tracing of Regenerated Axons
To evaluate whether the treatments with hMSC-seeded scaffolds enhances regeneration of damaged axonal projections through the lesion zone, animals were anesthetized as described above, and placedon Kopf stereotaxic frame. 4 weeks after the initial surgery, anterograde tracer biotinylated dextran amine (BDA; Molecular Probes, 10% wt/vol solution in PBS) was injected into the sensorimotor cortex (tracing for the corticospinal tract, CST) contralateral to the lesioned (spinal cord) side. Gelfoam cubes (1x1x1 mm) soaked in the retrograde tracer Fluorogold (FG, Fluorochrome Inc) (2% wt/vol solution in PBS) were inserted in two small incomplete lateral spinal cord myelotomies, which severed the axons but kept the lateral dura intact, 3cm caudal to the epicenter (Fig. 3).Figure 3. Neural tracing. Anterograde tracer biotinylated dextran amine (BDA) injection for corticospinal tract tracing and fluorogold (FG) injection for propriospinal interneuron: RN; red nucleus, SCI; spinal cord injury
The myelotomies were then sealed with Liquid BandaidTM (Johnson &
Johnson). Animals were maintained for 4 weeks before being sacrificed. BDA signal was revealed histochemically on floating or mounted 30µm spinal cord sections with a Vector Elite ABC kit (Vector Laboratories) and DAB kit (Pierce Biotechnology). Alternatively, Fluoroscein Avidin DCS (Vector Laboratories) was used to show BDA labeling under a fluorescent microscope.
Fluorogold signals were observed directly under a fluorescent microscope (Carl-Zeiss USA).
17.
Retrograde Tracing of Propriospinal Neuronal Projections
A subset of animals received intramuscular administration of fast blue (FB), a retrograde fluorescent tracer, to investigate propriospinal interneuron in the functional recovery of the animals. At 4 weeks post-injury the animals were re-anesthetized with the previously described protocol. The left latissimus dorsi and left intercostals muscles were exposed, with the proposed representation of the C7 and T7 nerve roots, respectively. FB (Polysciences), 1 µl of 2% solution was injected into 4 different locations within each muscle at an approximate depth of 2mm with a Hamilton syringe. The soft tissue was closed with suture and standard skin clips. The animals recovered and allowed to survive for 2 weeks, at which point they were euthanized and the spinal cords were explanted and prepared in the standard fashion for immunocytochemical analysis.
18.
Statistical Analysis
Unless otherwise specified, statistics were performed with SPSS software version 19 (IBM Corp, Somers, NY, USA). Comparisons of behavioral data, semiquntification of luminosity (for histopathology and ICC) as well as motor neuron quantifications between each treatment group was performed with one-way ANOVA testing with a 5% error and post-hoc Tukey’s HSD test.
Statistical significance was set at p < 0.05.
III. RESULTS
1. hMSC quality control
Given that most hMSC protocols involve multiple rounds of in vitro expansion to acquire sufficient cells for transplantation, especially for autologous transplantation, we considered it critical to first confirm that
“MSCness” was still maintained in our cells prior to use in any in vivo or in vitro studies. Even at passage 11 and 12, MSCs still expressed the characteristic markers CD90 and CD105 and possessed the capability of adipogenic, osteogenic, and chondrogenic differentiation as determined by Oil Red O, Alizarin Red S, and Alcian Blue staining, respectively, as well as real-time PCR determination of the mRNA expression of adiponectin (an adipogenic marker) and alkaline phosphatase (an osteogenic marker), although differentiability was reduced compared with passage 6 (Fig. 4).
I J Figure 4. . hMSC (human mesenchymal stem cells) quality control. A,B:
mmunostaining of hMSC markers CD105 (A) and CD90 (B) at passage 12. C,D:
Oil red O staining of adipogenic P12 (C) and P6 (D) hMSCs. E, F: Alizarin Red S staining of P11 hMSCs cultured in (E) control and (F) osteogenic medium. G,H:
Alcian Blue staining of (G) intact chondrogenic pellets and (H) pellet sections. I:
Real-time PCR to detect expression of adiponectin, an adipogenic marker, in P8 and P11 hMSCs cultured in adipogenic or control medium. J: Real-time PCR to detect expression of alkaline phosphatase (ALP), an osteogenic marker, in P8 and P11 hMSCs cultured in osteogenic or control medium.
Thus, given that differentiability is a reflection of “stemness” and our hypothesis that homeostatic modulation of the host environment is a prototypic progenitor cell characteristic, the MSCs in the proposed experiments were used between passages 5-7 to maximize their therapeutic effects.
2. Screening for neurotropic/neurotrophic effects of hMSCs
co-cultured with DRG explants
To investigate the paracrine effect of hMSCs on axon extension, we utilized the pseudounipolar morphology of the adult DRG neurons in the explants to evaluate the length of regenerated neurites in co-cultures with scaffolded hMSCs. Scaffolded hMSCs were co-embedded in matrigel 2 mm away from either the proximal or the distal axotomy site of each explanted DRG (Fig.5A &B). A non-seeded PLGA scaffold soaked with culture medium alone was co-embedded on the opposite side. This equally alternated arrangement allowed each DRG to serve as its own control. We observed a significant increase in the mean length of regenerated neurites (22% increase in average neurite length, p = 0.03) and in the mean distance away from the DRG that the neurites were able to extend (45% increase, p = 0.03) on the side of the DRG exposed to the scaffolded hMSCs when compared with the opposite side exposed to the control scaffold (Fig.5C &D). hMSCs in co-cultures and in scaffolds expressed bioactive molecules, e.g., BDNF and ciliary neurotrophic factor (CNTF), that have been suggested to mediate hMSC-derived therapeutic effects on neurological cells/tissue. Notably, the secretion of BDNF was context dependent, i.e., BDNF secretion by hMSCs increased in the presence of DRG tissue (Fig.5E &F)
Figure 5. Neuritogenic effects of hMSCs co-cultured with explanted adult DRGs. A,B: DRG co-cultured with hMSC-incorporated and non-seeded scaffolds facing the distal (A) and proximal (B) axotomy sites, respectively. C:
Regenerated neurite length. The “number of neurites” on the x-axis refers to the number of neurites averaged to provide the mean neurite length. The MSC + DRG condition showed a significant increase when considering 3, 5, and 10 neurites (p = 0.03, Mann-Whitney). D: Maximum growth cone extension. All
growth cones were growth cone extension distance for those DRGs. The “number of neurites” on the x-axis is as described for C. The MSC + DRG condition showed a significant increase per 10 or 15 neurites (p = 0.03, Mann-Whitney). E:
CNTF mRNA expression in scaffolded hMSCs cultured alone and co-cultured with DRG explants. F: Levels of secreted hBDNF in the supernatants of DRGs cultured individually or with hMSCs.
DRG: dorsal root ganglion, hMSC: human mesenchymal stem cell, CNTF: ciliary neurotrophic factor, BDNF: brain derived neurotrophic factor
3. Screening for anti-inflammatory effects of hMSCs co-cultured with DRG explants
For effective therapeutic approaches to neurotrauma, however, the prevention of neuroinflammation and the secondary injury process may be of equal or greater importance than axonal regeneration. To simulate post-SCI inflammatory pathology, we treated our co-culture system with bacterial LPS to induce an inflammatory response at 18 h after the initiation of co-culture. LPS has been previously used to induce acute inflammation in DRG neurons, Schwann cells, and microglia. Additionally, LPS injection into the spinal cord has been shown to locally upregulate TNF-α; this makes our model particularly relevant as TNF-α is an early pro-inflammatory marker in acute SCI. We first optimized the concentration of LPS to generate our model; we observed robust TNF upregulation upon the application of 10-20 ng/ml LPS (Fig. 6A). DRGs treated with 20 ng/ml LPS showed transient spikes of TNF, IL-1, and IL-6 expression reminiscent of that seen in vivo in the acute phase of neurotrauma
(Fig. 6B-D).
A
B C
D
Figure 6. Generation of lipopolysaccharide (LPS)-induced inflammation model in DRG explants. A: Adult rat DRGs were explanted and seeded in Matrigel and treated for 2 hours with LPS at different concentrations. Relative mRNA expression of TNF, an inflammatory marker, was quantified using real-time PCR. B,C,D: Rat DRG explants were treated for 0.5, 6, 12, or 24 hours with 2 ng/ml LPS. Relative mRNA expression for TNF (B), IL-1 (C), and IL-6 (D), characteristic inflammatory markers, was quantified using real-time PCR.
Co-culture with scaffolded hMSCs decreased DRG mRNA expression of the pro-inflammatory cytokines TNF-α and IL-1 by 62% and 65%, respectively (Fig. 7A &B). DRG expression of IL-6 was decreased by 72% (Fig. 6C).
A
B
C
Figure 7. Anti-inflammatory effects of hMSCs in DRG explants. DRG (dorsal root ganglion) and scaffolded hMSCs were co-cultured and treated with 10 ng/ml LPS (lipopolysccharide) for 2 hours. Fold difference in expression of rat inflammatory cytokine mRNA is expressed relative to naïve DRG. Co-culture with scaffolded hMSCs decrease DRG mRNA expression of TNF-α (A), IL-1 (B), and IL-6 (C).
4. Sensorimotor function:
SCI rats were quantitatively tested for open-field locomotion with the BBB scale. The mean BBB score for the hindlimb ipsilateral to the injury site in the scaffold+hMSC treatment group was significantly higher (p<0.05, 1-way ANOVA) throughout the 4 weeks after injury relative to all three control groups (Fig. 8A). The BBB score of the contralateral hindlimb of animals in the scaffold+hMSC group was also higher than that in the lesion alone group (data not shown). An inclined plane was used to test forelimb strength in the upward-facing orientation, which should be unaffected by this thoracic injury,
as well as coordinated hindlimb motor function in the downward-facing orientation10, 11. The treatment group achieved higher (downward facing) inclined plane angles than all three control groups (lesion only, hMSC only and scaffold only) (Fig. 8B). All animals also underwent weekly reflex testing and grading, where a score of 0 represented no response to the stimulus, 1 was a reduced response, 2 was a normal response, and 3 was a hyperactive response.
The data for the spinal reflexes are presented as the percentage of animals in each group (n=7/group) displaying a normal response (i.e., a score of 2; 71% of rats in the scaffold+hMSC group displayed a normal response to a brief pressure stimulus to the left hind limb at 4 weeks post SCI, whereas 100% of the lesion-alone and scaffold-alone control rats remained bilaterally hyper-reflexic at 4 weeks post-SCI. The withdrawal reflex testing demonstrated a similar trend in improvement. By 4 weeks post-injury, 57% of the animals in the scaffold+hMSC group demonstrated a normal righting reflex, whereas 0% of the lesion-alone, hMSC-alone and scaffold-alone control group rats had normal responses. These data suggest that the scaffold+hMSC treatment was beneficial in maintaining spinal-cord-mediated reflex responses in the SCI rats (Fig. 8C-E)
Dysethesias, allodynia and other sensory perturbations are common complaints of patients living with chronic SCI. However, the mechanisms and hence treatment of this pain is poorly understood and ineffectively treated12. We thus evaluated the effects of the proposed scaffold and stem cell-based treatments on not only motor function but also allodynia, i.e., nociceptive
responses to a stimulus that is normally not noxious. We conducted a barrage of sensory tests with standard 2g and 10g Semmes-Weinstein filaments. Each week, animals were tested with brief tactile stimulation from the filaments at the level of the injury (approximate dermatome), above and below the level, dorsally and ventrally, as well as in each paw. At 4 weeks post-SCI, the animals treated with scaffolded hMSCs demonstrated a significantly lower prevalence of allodynia relative to the lesion controls (Fig. 8F), which substantiates the benefit of the scaffold+hMSC therapy for the maintenance and recovery of not only motor but also sensory function.
A
B
C
D
E
F
Figure 8. Behavior assessments: Sensorimotor, coordination and spinal reflex tests through the first four weeks post-injury. A) Open field locomotion as measured by the BBB scoring paradigm B) Hindlimb coordination testing with the rat facing downward on an inclined plane, C & D)Pressure and pain withdrawal reflexes for the hindlimb ipsilateral to the injury side. E) Recovery in the contact righting reflex. F)Von Frey filament testing was performed with 2g and 10g filaments. At-level allodynia tests performed at 4 weeks post-injury.
5.
Histopathology and immunocytochemistry
:Spinal cord sections from animals of all groups were prepared for histopathologic evaluation with hematoxylin & eosin (H&E) and solvent blue. A qualitative analysis confirmed the complete degradation of the PLGA scaffold in vivo, which confirmed that the PLGA scaffold was biocompatible and biodegradable. The scaffold degradation was further investigated in a subsequent experiment. In a quantitative assessment (Fig. 9A &B) of the tissue
sparing in 3 representative spinal cords from each group (i.e., tissue from SCI rats with BBB scores closest to the group mean) the group treated with scaffolded hMSCs showed the most extensive tissue sparing: the mean lesion volume was 8.76 mm3 in the lesion-alone group as compared to 3.99 mm3 in the scaffold+hMSC group, which represents a 54% reduction in tissue degeneration (p<0.05). The scaffold+hMSC treatment group also tended to show an increase in the spared white matter relative to the means from the three control groups (Fig. 9C), although this increase did not reach statistical significance. To examine the differential sparing and/or sprouting of motor neurons in the anterior horns of the peri-epicenter spinal cord tissues, we manually identified and counted the motor neurons in 1-mm intervals rostral and caudal to the injury epicenter in the anterior horns of representative spinal cords from each group (H&E and solvent blue staining). Although the difference did not reach statistical significance, the absolute number of motor neurons caudal to the injury epicenter was higher in the spinal cords treated with scaffolded hMSCs than in the other groups, which supports the observed functional motor improvement in the treated animals (Fig. 9D).
A
B
C
D
Figure 9. Histopathological analyses: A) Hematoxylin & eosin and solvent blue staining of transverse sections from a representative spinal cord each study group.
B) Quantification of lesion volume and C) white matter sparing around the epicentre. The average total lesion volume in scaffold plus hMSCs group is significantly smaller than that in the other groups (p<o.o5, one way ANOVA). D) Quantification of motor neurons in the ventral horns of the injured spinal cord demonstrates that the average number of surviving motor neurons is higher in the scaffold plus hMSCs group than that in the other groups.
Immunocytochemistry was performed to characterize the molecular mechanisms potentially underlying the functional benefits of the scaffold and hMSC constructs. Endogenous stem cell proliferation was significantly increased (p<0.05) in the scaffold+hMSC spinal cords relative to all three control conditions as measured by a 314% semi-quantitative increase in doublecortin staining (DCX; Fig. 10A) in the scaffold-hMSC group relative to the lesion-alone group. The enhanced proliferation of endogenous neural stem cells was also supported by immunostaining for rat nestin (data not shown). The
activation of endogenous stem cells by hMSCs has been previously demonstrated13; however, the large difference compared with animals treated with hMSCs alone animals suggests that the hMSCs were held in place by the scaffold to effectively influence endogenous neural progenitors, rather than being washed away in the interstitial and cerebrospinal fluid. The hMSC+scaffold treatment also provided increased angiogenesis; staining for laminin, an angiogenic marker, was nearly four-fold higher (p<0.05) than in lesion-alone group (Fig. 10B). Improved angiogenesis within and surrounding the injury epicenter should enhance the healing of the spinal cord and may contribute to the overall functional benefit of the combined construct.
The inflammatory cascade following spinal cord injury is complex and both contribute to secondary neural damage as well as regeneration14,15. Immunomodulation has become a centerpiece to approaches for neuronal regeneration after CNS injury. We used hMSCs inherent ability to home to sites of injury and engender an environment to limit the deleterious effects of obligatory inflammation after SCI. Immunohistochemistry was also used to test the hypothesis that secondary injury and tissue damage were decreased after treatment with scaffolded hMSCs. Inflammatory markers were evaluated in transverse sections at and adjacent to the injury epicenter to characterize the acute and chronic anti-inflammatory effects of the treatment. The immunoreactivity for GFAP, a marker of astroglial scarring, was lowest in tissue from animals treated with scaffold hMSCs (Fig. 10C). The immunoreactivity
for nitrotyrosine (Fig. 10D), which is a marker of protein nitration that indicates oxidative damage resulting from reactive nitrogen or oxygen species, was reduced in spinal cords from animals treated with scaffolded hMSCs. The immunoreactivity for CD68 (Fig. 10E) and CD11b (not shown), which are markers of activated microglia/macrophages, was decreased in spinal cords treated with scaffolded hMSCs (p<0.05 against all other conditions), thereby indicating an anti-inflammatory effect against cells that may be involved in the evolution of secondary neural loss and degeneration following SCI.
Immunohistochemistry was also used to evaluate the expression of potential molecular mediators of hMSC-derived effects. BDNF expression was increased by two to three-fold (p<0.05) in the spinal cords of the animals receiving scaffolded hMSCs, and co-staining for DAPI and HSP27 confirmed that the BDNF expression was co-localized with the remaining implanted hMSCs.
Expression of IL-10, which is a cytokine that is primarily thought to have anti-inflammatory effects, was significantly higher in the scaffold + hMSC condition (p<0.05) than in any of the other conditions. Co-staining with DAPI and CD90, which is another hMSC marker), confirmed that the IL-10 was
Expression of IL-10, which is a cytokine that is primarily thought to have anti-inflammatory effects, was significantly higher in the scaffold + hMSC condition (p<0.05) than in any of the other conditions. Co-staining with DAPI and CD90, which is another hMSC marker), confirmed that the IL-10 was