II. MATERIALS AND METHODS
5. Immunocytology for co-cultures
Co-cultures were fixed in 4% paraformaldehyde in 0.1M phosphate buffer at 37 ºC for 3 hours. After washing in PBS, co-cultures were blocked for 1 hour at room temperature in PBS supplemented with 2% donkey serum. They were then incubated overnight at room temperature with primary antibodies against ß3-tubulin (1:300), human heat shock protein (HSP) 27 (1:300), or BDNF (1:300). After washing in PBS, incubation with FITC- and Texas Red-labeled secondary antibodies (1:200) was performed at room temperature for 3 hours. Co-cultures were then immediately imaged after the final PBS wash while still immersed in PBS.
6.
Imaging
All imaging was performed on a Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) equipped with a Spot RT-Slider CCD camera (Diagnostic Instruments, Sterling, MI) or a Zeiss Axiovert 200 microscope (Zeiss) equipped with an Axiocam CCD camera (Zeiss) using appropriate filters for FITC, Texas Red, and DAPI when necessary.
7.
Quantification of Neurite Turning Behavior and Neurite Length
Neurite turning behavior was quantified and the “measure angle”function of ImageJ software (NIH, USA) was used to evaluate the angle between the direction of neurite extension at the point of emergence from the DRG and the point of termination at the neurite tip. Thus, neurite extension in a
straight line would result in an angle measurement of 0º. Neurites were only measured if the point of origin and point of termination were clearly visible.
Neurite length was measured using the “segmented line” function of ImageJ software (NIH, USA) and defined as the length along the neurite from the point of emergence from the DRG to the point of termination at the neurite tip. Again, neurites were only measured if the point of origin and point of termination were clearly visible. This measurement of neurite length accounted for the tortuosity of the neurites; i.e., if two neurites extended out the same total distance from the DRG, the neurite that followed the straightest path without turning or switching back on itself would return a lower value. To account for the total extension distance, i.e., how far from the DRG the neurite was able to extend, we also measured the maximum extension distance as the absolute distance from the point of emergence of the DRG to the point of termination at the neurite tip, regardless of the path followed by the neurite or its tortuosity.
8.
BDNF Enzyme-linked Immunosorbent Assay
Supernatants were collected from co-cultures, snap-frozen in liquid nitrogen, and stored at -80 ºC. TNF-α and BDNF were quantified using enzyme-linked immunosorbent assay kits (R&D Systems, Inc. Minneapolis, MN) according to the manufacturer’s instructions with minor modifications.
Optical density was measured at a wavelength of 450 nm and a reference wavelength of 570 nm. Density values were correlated linearly with the
concentrations of cytokine standards.
9.
Real-time PCR
As various times after the application of LPS, the medium was removed from the well plates, and the plates were frozen at -80 ºC. While still frozen, DRGs and scaffolded hMSCs were removed from the well plates using forceps and placed in the lysis buffer of the RNAqueous kit (Ambion) for total RNA extraction; extraction then proceeded per the instructions of the manufacturer and the extracted RNA was concentrated to 15 µl using sodium acetate and linear acrylamide per the instruction of the manufacturer. The RNA was treated with DNase per the instructions of the manufacturer to prevent contamination by genomic DNA. Reverse transcription was then performed using the Superscript III kit (Invitrogen) per the instructions of the manufacturer.
Real-time PCR was performed using the 2X SybrGreen master mix (Applied Biosystems) per the instructions of the manufacturer on an Applied Biosystems 7900 HT Fast Real Time PCR system (California, USA), with a dissociation curve analysis performed to confirm the specificity of the reaction and lack of primer dimerization. PCR primers were selected from previous reports or designed using PrimerExpress software. Relative quantification of the amplification data was performed using the Ct method.
10.
Spinal Cord Injury and Animal Care
Surgeries were performed in a block design, and treatments were determined in a random fashion after the spinal cord injury was completed9. Female Spague-Dawley (SD) rats were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) and placed prone and a 3cm midline incision was made over the lower thoracic spine after standard sterile preparation of the skin. Soft tissue dissection was followed by laminectomies at the 9th and 10th thoracic vertebral levels (T9-10). A No. 11-blade scalpel was used to make a midline myelotomy and transverse hemisections were carried out to complete a unilateral (left-sided) segmental hemisection, measuring 4mm rostral-caudal.
Hemostasis was achieved and either PLGA scaffold seeded with hMSCs, PGLA scaffold alone, hMSCs alone or saline (i.e. lesion control) were placed into the hemisection cavity (Fig. 1).
A B C
Figure 1. Injury model and scaffold design. A) unilateral hemisection by creating 4 mm longitudinal cut, B & C) implantation of PLGA (poly-lactic-co-glycolic acid) scaffold seeded with human mesenchymal stem cells (hMSCs).
There were n=7 animals for each of the 4 groups. Following implantation of the treatment, the spinal musculature and fascia was
approximated and closed with a 4-0 non-absorbable suture. The skin was closed with wound clips in a standard fashion and animals were administered daily Lactated Ringer’s and ketoprofen subcutaneously for 5 days. Bladders were manually expressed twice daily for 5 days following SCI or until normal bladder reflexes returned. To sufficiently immunosuppress the animals treated with hMSCs, all (both treatment and control groups) were administered tacrolimus (1mg/kg) daily for 2 days prior to surgery and daily after surgery until perfusion. Animal procedures were conducted in accordance with the CHA University Institutional Animal Care and Use Committee (IACUC).
11.
Behavior Evaluation
Behavioral analysis was conducted by two observers, blinded to the treatment identity. Coordinated motor activity was evaluated using the Basso, Beattie and Bresnahan (BBB) locomotor rating scale (scored on a 21 point scale)28. Ability to maintain stable body position was performed with an incline plane. For the inclined plane test, the highest degree of inclination was defined as being that at which the animal could maintain its position for 5 seconds on two separate trials11. Reflexes including toe spread, withdrawal reflexes to extension, pain and pressure, as well as placing and righting were also performed and graded as described by Gale10. Animals were tested on the first postoperative day and weekly thereafter.
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,
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,