Label-free multiphoton microscopy reveals relevant tissue changes induced by alginate hydrogel implantation in rat spinal cord injury

Roberta Galli,Edmund Koch,Ortrud Uckermann,Maria Koch,Kerim H Sitoci-Ficici,Gerald Steiner,Elke Leipnitz,Robert Later,Matthias Kirsch,Gabriele Schackert,Magda Marečková,Michael Gelinsky

doi:10.1038/s41598-018-29140-z

Roberta Galli, Edmund Koch + Show 10 more

Open Access

https://doi.org/10.1038/s41598-018-29140-z

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Abstract

The development of therapies promoting recovery after spinal cord injury is a challenge. Alginate hydrogels offer the possibility to develop biocompatible implants with mechanical properties tailored to the nervous tissue, which could provide a permissive environment for tissue repair. Here, the effects of non-functionalized soft calcium alginate hydrogel were investigated in a rat model of thoracic spinal cord hemisection and compared to lesioned untreated controls. Open field locomotion tests were employed to evaluate functional recovery. Tissue analysis was performed with label-free multiphoton microscopy using a multimodal approach that combines coherent anti-Stokes Raman scattering to visualize axonal structures, two-photon fluorescence to visualize inflammation, second harmonic generation to visualize collagenous scarring. Treated animals recovered hindlimb function significantly better than controls. Multiphoton microscopy revealed that the implant influenced the injury-induced tissue response, leading to decreased inflammation, reduced scarring with different morphology and increased presence of axons. Demyelination of contralateral white matter near the lesion was prevented. Reduced chronic inflammation and increased amount of axons in the lesion correlated with improved hindlimb functions, being thus relevant for locomotion recovery. In conclusion, non-functionalized hydrogel improved functional outcome after spinal cord injury in rats. Furthermore, label-free multiphoton microscopy qualified as suitable technique for regeneration studies.

Highlights

Every year, between 250,000 and 500,000 people around the world suffer a spinal cord injury (SCI)[1]
A soft calcium alginate hydrogel preparation led to implants that remained stable in a rat model of SCI up to six months after injury[24,25], with positive effects on fibrotic scarring and contralateral demyelination[25]
The hindlimb function was tested in open field from 3 days post-injury (DPI) until the chronic phase at 154 DPI (Fig. 1a)

Summary

Introduction

Between 250,000 and 500,000 people around the world suffer a spinal cord injury (SCI)[1]. We demonstrated that soft alginate hydrogels prepared with sub-stoichiometric concentration of Ca2+ cations support neurite growth in vitro and protect neuronal cells against oxidative stress without the need of functionalization[18]. Implantation of such alginate hydrogels in small spinal cord lesions in rats improved functional recovery[22]. Second harmonic generation (SHG) visualizes collagen type I34 The combination of these three multiphoton imaging techniques enables a comprehensive study of SCI35. The technique is well suited for observation of small details and delicate tissue structures that might be lost during conventional staining procedures

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Conclusion