Neural stem cell mediated recovery is enhanced by Chondroitinase ABC pretreatment in chronic cervical spinal cord injury

<div><p>Traumatic spinal cord injuries (SCIs) affect millions of people worldwide; the majority of whom are in the chronic phase of their injury. Unfortunately, most current treatments target the acute/subacute injury phase as the microenvironment of chronically injured cord consists of a well-established glial scar with inhibitory chondroitin sulfate proteoglycans (CSPGs) which acts as a potent barrier to regeneration. It has been shown that CSPGs can be degraded <i>in vivo</i> by intrathecal Chondroitinase ABC (ChABC) to produce a more permissive environment for regeneration by endogenous cells or transplanted neural stem cells (NSCs) in the subacute phase of injury. Using a translationally-relevant clip-contusion model of cervical spinal cord injury in mice we sought to determine if ChABC pretreatment could modify the harsh chronic microenvironment to enhance subsequent regeneration by induced pluripotent stem cell-derived NSCs (iPS-NSC). Seven weeks after injury—during the chronic phase—we delivered ChABC by intrathecal osmotic pump for one week followed by intraparenchymal iPS-NSC transplant rostral and caudal to the injury epicenter. ChABC administration reduced chronic-injury scar and resulted in significantly improved iPSC-NSC survival with clear differentiation into all three neuroglial lineages. Neurons derived from transplanted cells also formed functional synapses with host circuits on patch clamp analysis. Furthermore, the combined treatment led to recovery in key functional muscle groups including forelimb grip strength and measures of forelimb/hindlimb locomotion assessed by Catwalk. This represents important proof-of-concept data that the chronically injured spinal cord can be ‘unlocked’ by ChABC pretreatment to produce a microenvironment conducive to regenerative iPS-NSC therapy.</p></div

ChABC treatment reduces components of glial scar including astrocytes and chondroitin sulfate proteoglycans.

<p>Representative longitudinal and axial sections immunostained for CSPGs (CS56⁺) and astrocytes (GFAP⁺). (A, C) Extensive CSPG deposition and astrogliosis are found 16 weeks after injury in vehicle treated animals, however, (B, D) ChABC treatment 7 weeks after injury results in sustained reductions in CSPG and astrocyte labelling at 16 weeks post-injury. (E, F) Fold decreasing in GFAP and CS56 immunointensity in three longitudinal sections in each group (n = 2 per group). ChABC treated groups show a decrease in the total labelling area compared to the SCI group, however, statistical analysis was not performed because of the small number of animals. Scale bars = 500 μm (A-D).</p

Schematic representation of experimental design.

<p>Treatments are in green; assessments are in blue. (A) A C6 clip-contusion spinal cord injury (SCI) was performed at 0 weeks in C57/BL6 mice. Seven weeks post-injury, a mini-osmotic pump was implanted rostral to the injury site. The pump delivered either Chondroitinase ABC (ChABC) in artificial cerebral spinal fluid (aCSF) or aCSF alone. One week later the pump was explanted and induced pluripotent stem cell-derived neural stem cells (iPS-NSC) were transplanted into the cord parenchyma at two rostral and two caudal sites. 50,000 cells in 1μL were delivered to each site. Cyclosporine A immunosuppression was delivered from 2 days prior to transplantation until the end of the study. Behavioral assessments were performed across all 16 weeks of the study. At 16 weeks electrophysiologic assessments were completed followed by whole-animal perfusion, fixation and immunohistochemical analyses. (B) Outline of the control and treatment groups. For additional details please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182339#pone.0182339.s005" target="_blank">S1 Table</a>.</p

iPS-NSC differentiate along all three neuroglial lineages after transplant into ChABC pretreated chronically injured spinal cords.

<p>(A) Immunofluorescence imaging demonstrating undifferentiated neural stem cells (NES⁺/GFP⁺), neurons (NeuN⁺/GFP⁺), astrocytes (GFAP⁺/GFP⁺), and oligodendrocytes (APC⁺/GFP⁺) indicated by white arrowheads. Scale bar represents 50μm. (B) Quantification of GFP⁺ differentiated cell counts using optical fractionation and unbiased stereological techniques. iPS-NSC transplanted into aCSF (control) and ChABC pretreated cords demonstrated similar proportions of cell differentiation. Cell counting for immunofluorescent-positive cell shows a similar differentiation pattern for iPS-NSC with and without ChABC pretreatment. Data represent mean ± SEM, n = 6 per group.</p

Patch-clamped transplanted GFP⁺ cells in <i>ex vivo</i> cords demonstrate functional action potentials.

<p>(A) Electrical tracing demonstrating currents evoked by voltage pulses which contain transient inward sodium currents and outward potassium currents. Membrane potential was held at -80 mV and depolarized to +30 mV with an increment of 10 mV step (48 ms long). (B) Representative spontaneous postsynaptic currents with burst and sporadic firing patterns. (C) Time expanded tracings of key (i) burst and (ii) sporadic postsynaptic currents in B. (ii) Two types of spontaneous postsynaptic currents are seen with slow decay time and (*) fast decay time. Membrane potential was held at -80 mV in B and C. n = 4 animals and 12 GFP⁺ cells per group. iPS-NSC transplant enhances recovery of in vivo motor evoked potentials (MEPs). MEPs were stimulated at the C2 cervical spinal cord and recorded from the hypothenar muscles. (D) At 16 weeks post-SCI, only mice treated with iPS-NSCs had significantly shorter MEP latencies while (E) higher peak MEP amplitudes were found in both the iPS-NSC and ChABC + iPS-NSC treated groups. Data are mean ± SEM values, n = 6 per group. (*) indicates statistical significance at p<0.05</p

ChABC pretreatment enhanced transplanted iPSC-NSC survival.

<p>(A,B,C) Representative longitudinal and axial sections of injured spinal cords 8 weeks after transplant (16 weeks after injury). Scale bar represents 200 μm in A and B. Scale bar represents 50 μm in C. A greater number of GFP⁺ iPS-NSC can be seen throughout the ChABC pretreated cord than the control cord. (D) Quantification of GFP⁺ cell counts using optical fractionation and unbiased stereological techniques. GFP⁺ iPS-NSC survival was significantly higher in the ChABC pretreated group by more than 3 times. (E) Increased survival was seen in sections at the epicenter and rostrally/caudally up to 1440μm. Data represented as mean ± SEM; n = 6 per group. (*) indicates statistical significance at p<0.05</p

Electrophysiological assessment following AdV-ZFP-VEGF administration.

<p>(A) Representative tracings of MEP's recorded from the hindlimb at 8 weeks post-injury. (B) MEP quantification. Recordings were obtained from hindlimb biceps femoris. Stimulation was applied to the midline of the cervical spinal cord (0.13 Hz; 0.1 ms; 2 mA; 200 sweeps). Latency was calculated as the time from the start of the stimulus artifact to the first prominent peak. AdV-ZFP-VEGF did not result in improved MEP's. (C) H-Reflex quantification. Recording electrodes were placed two centimeters apart in the mid-calf region and the posterior tibial nerve was stimulated in the popliteal fossa using a 0.1 ms duration square wave pulse at a frequency of 1 Hz. The rats were tested for maximal plantar H-reflex/maximal plantar M-response (H/M) ratios to determine the excitability of the reflex. AdV-ZFP-VEGF administration did not significantly alter the H/M ratio. n = 6/group.</p

Tissue sparing quantification at 8 weeks post-SCI.

<p>(A) Residual white matter quantification. (B) Residual grey matter quantification. AdV-ZFP-VEGF improves spinal cord grey matter preservation. (C) Representative sections are shown from each group. Sections shown are taken 2 mm rostral to the epicenter at 8 weeks after SCI. AdV-ZFP-VEGF treated spinal cord exhibited a larger extent of grey matter spared tissue, but not white matter; **p<0.001. Data are mean ± SEM values. n = 8/sham and AdV-ZFP-VEGF groups; n = 10/injured control and AdV-eGFP groups.</p

AdV-ZFP-VEGF administration attenuated axonal degradation and increased neuron sparing.

<p>(A) Western blot indicates that administration of AdV-ZFP-VEGF resulted in a significant attenuation of NF200 degradation 10 days after injury. Lower panel shows actin protein control. (B) Relative OD value of controls versus AdV-ZFP-VEGF treated animals. Significant NF200 sparing was observed in AdV-ZFP-VEGF-treated animals compared to control groups at 10 days after injury, although all injured groups showed significant NF200 loss following SCI. Optical density of NF200 was normalized to actin. One-way ANOVA (Holm-Sidak post-hoc), *p<0.05. (C) Representative sections taken 2 mm rostral to the epicenter from AdV-ZFP-VEGF treated and AdV-eGFP treated animals immunostained with NeuN at 5 days after SCI; scale 200 µm. A greater number of NeuN-positive cells were observed in animals treated with AdV-ZFP-VEGF. (D) Bar graph shows quantification of the NeuN-positive cell counts at 5 days after SCI. There was a significant preservation of neurons overall in the AdV-ZFP-VEGF group compared to all the other injured groups (two-way ANOVA comparing distance from the epicenter and treatment group). Bar graph shows mean OD values ± SEM. Two-way ANOVA (Holm-Sidak post-hoc), *p<0.02. n = 5/sham, n = 4/injured control, AdV-eGFP and AdV-ZFP-VEGF groups.</p

AdV-ZFP-VEGF improves hindlimb weight support.

<p>Catwalk gait analysis was used to assess hindlimb weight support. A sub-set of animals (with BBB scores >9) were assessed every week between 4–8 weeks, and each animal performed a standardized Catwalk run. A blinded observer analyzed the data. (A) Paw area: the maximal area of the paw print in contact with the detection surface of the CatWalk (expressed in mm²), (B) Paw width: the maximal distance spanning the medial and lateral contact points of the paw (expressed in mm), and (C) Paw length: the maximal distance spanning the cranial and caudal contact points of the paw (expressed in mm). (D) Representative images of CatWalk forelimb (green) and hindlimb (red) prints, which were used to quantify the data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096137#pone-0096137-g006" target="_blank">Figure 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096137#pone-0096137-g007" target="_blank">Figure 7</a>. Data presented is the mean ± SEM, n = 5/group, at 8 weeks following SCI. One-way ANOVA (Holm-Sidak post-hoc). *p<0.05, **p<0.005.</p