Inflammation-related alterations of lipids after spinal cord injury revealed by Raman spectroscopy

Spinal cord injury (SCI) triggers several lipid alterations in nervous tissue. It is characterized by extensive demyelination and the inflammatory response leads to accumulation of activated microglia/macrophages, which often transform into foam cells by accumulation of lipid droplets after engulfment of the damaged myelin sheaths. Using an experimental rat model, Raman microspectroscopy was applied to retrieve the modifications of the lipid distribution following SCI. Coherent anti-Stokes Raman scattering (CARS) and endogenous two-photon fluorescence (TPEF) microscopies were used for the detection of lipid-laden inflammatory cells. The Raman mapping of CH2 deformation mode intensity at 1440 cm−1 retrieved the lipid-depleted injury core. Preserved white matter and inflammatory regions with myelin fragmentation and foam cells were localized by specifically addressing the distribution of esterified lipids, i.e., by mapping the intensity of the carbonyl Raman band at 1743 cm−1, and were in agreement with CARS/TPEF microscopy. Principal component analysis revealed that the inflammatory regions are notably rich in saturated fatty acids. Therefore, Raman spectroscopy enabled to specifically detect inflammation after SCI and myelin degradation products

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

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

Biochemical Monitoring of Spinal Cord Injury by FT-IR Spectroscopy--Effects of Therapeutic Alginate Implant in Rat Models.

Spinal cord injury (SCI) induces complex biochemical changes, which result in inhibition of nervous tissue regeneration abilities. In this study, Fourier-transform infrared (FT-IR) spectroscopy was applied to assess the outcomes of implants made of a novel type of non-functionalized soft calcium alginate hydrogel in a rat model of spinal cord hemisection (n = 28). Using FT-IR spectroscopic imaging, we evaluated the stability of the implants and the effects on morphology and biochemistry of the injured tissue one and six months after injury. A semi-quantitative evaluation of the distribution of lipids and collagen showed that alginate significantly reduced injury-induced demyelination of the contralateral white matter and fibrotic scarring in the chronic state after SCI. The spectral information enabled to detect and localize the alginate hydrogel at the lesion site and proved its long-term persistence in vivo. These findings demonstrate a positive impact of alginate hydrogel on recovery after SCI and prove FT-IR spectroscopic imaging as alternative method to evaluate and optimize future SCI repair strategies

Analysis of the fibrotic lesion.

<p>(A) Representative IR spectra of the fibrous scar six months after injury, of grey matter and of reference collagen. (B) Spectroscopic images showing the intensity of the band 1242 cm^-1 in pseudo color. They depict the distribution of collagen in control and alginate-implanted samples one and six months post-injury. Scale bar: 0.5 mm. (C) Fibrous scar thickness of control and alginate-implanted samples at one and six months post-injury, n = 5–7, two-tailed t-test, *: p < 0.05.</p

Distribution of alginate within the tissue six months after injury.

<p>(A) spectroscopic RGB image, generated by combining the intensity of bands at 1420 cm^-1 (alginate hydrogel–red), 1653 cm^-1 (nervous tissue–green) and 1242 cm^-1 (collagen—blue). (B) Alcian blue staining of a consecutive section. Scale bars: 100 μm. The boxes in A and B indicate the area of magnification. The arrowheads in the magnifications indicate small inclusions of alginate hydrogel into the tissue that colocalize in the spectroscopic image and in the staining. The asterisks indicate alginate inside large cysts.</p

Analysis of the contralateral nervous tissue.

<p>(A) Spectroscopic images showing the intensity of the band at 1735 cm^-1 (ν_s(C = O)). They depict the distribution of lipids in control and alginate-implanted samples one and six months post-injury. Scale bar: 1 mm. (B) Intensities of the bands at 1735 cm^-1, 1466 cm^-1 (δ[(CH₂)], also showing the distribution of lipids) and 1225 cm^-1 (ν_as(PO₂⁻), showing the distribution of phospholipids) in the white matter contralateral to the lesion indicated by the black boxes in panel A normalized for each samples to the intensity in the region indicated by white boxes in panel A. For each group n = 5–6. Two-tailed t-test, *: p < 0.05, **: p < 0.01.</p