Adipose-derived stem cell conditioned medium impacts asymptomatic peripheral neuromuscular denervation in the mutant superoxide dismutase (G93A) transgenic mouse model of amyotrophic lateral sclerosis

2.1 Animals

We utilized female mSOD1^G93A ALS mice [B6SJL-Tg(SOD1*G93A)1Gur/J; Jackson Laboratories] (n = 16). Female wild-type (WT) mice served as controls (Jackson Laboratories, n = 9). For time course assessment of NMJ innervation, mSOD1^G93A mice were euthanized with an overdose of ketamine/xylazine at post-natal day 35, 39, 43 and 47 (n = 3-4/timepoint). WT mice were euthanized at post-natal day 47 (n = 3). Once anesthetized, as determined by lack of response to toe pinch stimulus, transcardial perfusion was performed with 50 ml phosphate buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M PBS for tissue fixation. Once perfused, hindlimb gastrocnemius muscles were dissected and post-fixed for 40 min followed by transfer to 30% sucrose solution for cryoprotection and cryosectioning. In animals used for comparing ASC-CM therapy effects on NMJ innervation, mSOD1^G93A mice (n = 3-4) were treated with ASC-CM or Basal Medium Eagle (BME) and the mice were euthanized and tissue prepared as described above at post-natal day 47. All use of animals in this study followed the Guidelines for the Institutional Care and Use of Animals of Indiana University School of Medicine.

2.2 ASC-CM preparation and administration

Human subcutaneous adipose tissue samples were obtained from lipoaspiration/liposuction procedures and prepared as previously described (Gu et al., 2013; Traktuev et al., 2008; Wang et al., 2014). In brief, human adipose tissue samples were digested in collagenase type I(Worthington Biochemical) for 1 hr at 37°C, filtered with 100μm and 70μm filters, and centrifuged at 300 g for 5 min to separate stromal cells from adipocytes. The ASC pellet was treated with red blood cell lysis buffer for 5 min at 37°C, then centrifuged at 300 g for 5 min. The supernatant was discarded, and the cell pellet was resuspended in endothelial growth medium-2 MV (EGM-2-MV, Lonza) consisting of endothelial basal medium-2 (EBM-2), 5% fetal bovine serum (FBS), and the supplemental growth factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF-1). ASCs (4×10⁶ cells/cm²) were plated in T75 tissue culture flasks and incubated at 37°C in 5% CO₂. Fresh EGM2-MV was replaced the next day. ASCs were cultured to 80% confluence before passaging and freezing. Passage 4 ASCs were seeded at 5×10⁴ cells/cm² and grown to 80% confluence, and then rinsed and each T75 flask was incubated with 10 ml of Basal Media Eagle (BME) containing 5 mM KCl for 24 hrs. Conditioned media was collected, centrifuged for 5 min to eliminate debris and then filtered through a 22 μm micrometer sterile filter before freezing CM and BME in 1 ml aliquots for use in the study. Collection of human adipose tissue was approved by the Indiana University Institutional Review Board.

For treatment, mSOD1^G93A mice were randomly assigned to ASC-CM or vehicle treatment groups, and daily i.p. injections of 200μl ASC-CM or vehicle (Basal Medium Eagle, BME) were administered. Injections were started post-natal day 35 and continued to day 47. During treatment, the general health and overall behavior of the mice was regularly monitored by the investigators, animal facility veterinary staff and qualified veterinarians. Daily ASC-CM injections did not cause any observable outward physical or behavior changes in the mice or weight changes compared to vehicle treated mice during the course of the study.

2.3 Tissue preparation

Following perfusion, bilateral gastrocnemius and soleus muscles were dissected and prepared for cryosectioning. Following cryoprotection, right medial gastrocnemius muscle was isolated and placed in Tissue Tek OCT medium and frozen at –80°C. Embedded frozen muscle tissue was transferred to a cryostat chamber before cryosectioning. Prepared gastrocnemius tissue was serially sectioned at 20μm for immunofluorescent labeling of neuromuscular junctions and innervation.

2.4 Microscopic imaging and NMJ analysis

In brief, 8 serial sections separated by an interval of 100μm were air-dried on Superfrost microscope slides (Fisher Scientific), and incubated in donkey serum-based blocking buffer for 1 hr at room temperature. Next, the slides were incubated with primary antibodies against neurofilament (chicken anti-neurofilament, 1:500, Aves Inc.), which labels innervation, or synaptophysin (Alex 488-conjugated mouse anti-synaptophysin, 1:100, Millipore), which labels the neuromuscular pre-synapse, overnightat 4°C.

Slides were then washed in PBS+0.1% Triton-X100 (PBST). The slides incubated with primary neurofilament antibody were then incubated with donkey anti-chicken Alex 488-conjugated secondary antibody (Jackson Immunoresearch) and Alex 555-conjugated α-bungarotoxin (1:800, Life Technologies), which labels the post-synaptic acetylcholine receptors (AchRs) of the NMJ, for 1 hr at room temperature. The slides incubated with Alex 488-conjugated primary antibody against synaptophysin were also incubated with Alex 555-conjugated α-bungarotoxin (1:800, Life Technologies) for 1 hr at room temperature. Afterward, all slides were prepared for microscopic fluorescent imaging. Five random high-power fields per section were selected for quantification of synaptophysin/α-bungarotoxin (4 sections) and neurofilament/α-bungarotoxin co-labeled NMJs (4 sections). Quantification of percent intact NMJs (yellow) was determined by an overlay of α-bungarotoxin (red) and synaptophysin (green) or neurofilament (green). Denervated NMJs were defined by the presence of only α-bungarotoxin (red). Percent intact NMJs was calculated using the following formula: [number of intact NMJs (yellow) / total number of motor end plates (yellow + red)]×100. All slides were coded by an uninvolved investigator prior to analysis. Imaging was performed on an Olympus BX40 epifluorescent microscope and NMJ quantification was accomplished using NIH ImageJ software.

2.5 Statistical analysis

Statistical analysis was accomplished via an ANOVA and the Student-Newman-Keuls post hoc test (Sokal & Rohlf, 1995) using GraphPad Prism 6.0 software (GraphPad, Inc).

3.1 Gastrocnemius NMJ innervation significantly diminished between post-natal days 39 and 47 in mSOD1^G93A mice

Upon quantifying intact NMJs in the medial gastrocnemius of mSOD1^G93A and WT mice, we found that a significant decrease in neurofilament/α-bungarotoxin co-localized NMJs occurred between post-natal days 35 and 47 (p < 0.05) and days 39 and 47 in mSOD1^G93A mice (p < 0.05, Fig. 1A). The results from this time course suggest this decrease is rapid in mSOD1^G93A mice.

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Fig.1

NMJ denervation time course and therapeutic effects of ASC-CM on NMJ loss. A) Quantification of neurofilament and α-bungarotoxin NMJs showed significant reduction in co-labeling between post-natal days 35 and 47, and post-natal days 39 and 47 (p < 0.05). B & D) At post-natal day 47, mSOD1^G93A mice that were treated with daily i.p. vehicle showed significant loss of innervated NMJs as determined by neurofilament/α-bungarotoxin co-labeling compared to WT mice (p < 0.001). mSOD1^G93A mice treated with ASC-CM showed significantly more co-labeled NMJs than vehicle treated mice (p < 0.05) at this time point. C & E) Similar results were observed for pre-synaptic synaptophysin co-labeling with α-bungarotoxin. Significantly fewer co-labeled NMJs were observed in vehicle treated mSOD1^G93A mice than WT mice at post-natal day 47 (p < 0.01), while more co-labeling was observed with ASC-CM treatment compared to vehicle treatment (p < 0.05). ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001.

As our data showed intact NMJs were significantly decreased by post-natal day 47, we examined the effects of daily systemic ASC-CM therapy before and through this time period in mSOD1^G93A mice. When ASC-CM or BME vehicle was administered daily between post-natal days 35 and 47, ASC-CM treated mSOD1^G93A mice had significantly more neurofilament/α-bungarotoxin co-localized NMJs than vehicle treated mice, with co-localization rescued to WT levels (p < 0.05, Fig. 1B & D.) Likewise, synaptophysin-positive NMJs were significantly increased over vehicle treated mSOD1^G93A mice at post-natal day 47 (p < 0.05, Fig. 1C & E). Images shown in Fig. 1D and E are representative of the reduced intact NMJs following vehicle treatment, and increased intact NMJs observed through both neurofilament and synaptophysin co-labeling with post-synaptic AchRs following ASC-CM therapy.