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

Abstract

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.

Fig 1. IR spectroscopic imaging of SCI in rat models with and without alginate hydrogel implant at one and six months after injury.

(A) Spectroscopic images of longitudinal cryosections of the investigated samples, showing the intensity of the amide I band at 1653 cm^-1; asterisks (*) indicate large cysts. Scale bar: 1 mm. (B) Representative IR spectra of white and grey matter.

Fig 2. Analysis of the contralateral nervous tissue.

(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.

Fig 3. Analysis of the fibrotic lesion.

(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.

Fig 4. FT-IR spectroscopy of the alginate hydrogel implants.

IR spectrum of pure alginate hydrogel before its implantation (black) compared with the spectrum of alginate hydrogel in spinal cord tissue at one (red) and six months after injury (blue).

Fig 5. Distribution of alginate within the tissue six months after injury.

(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.