Innovative mouse model mimicking human-like features of spinal cord injury: efficacy of Docosahexaenoic acid on acute and chronic phases

Abstract

Traumatic spinal cord injury has dramatic consequences and a huge social impact. We propose a new mouse model of spinal trauma that induces a complete paralysis of hindlimbs, still observable 30 days after injury. The contusion, performed without laminectomy and deriving from the pressure exerted directly on the bone, mimics more closely many features of spinal injury in humans. Spinal cord was injured at thoracic level 10 (T10) in adult anesthetized female CD1 mice, mounted on stereotaxic apparatus and connected to a precision impactor device. Following severe injury, we evaluated motor and sensory functions, and histological/morphological features of spinal tissue at different time points. Moreover, we studied the effects of early and subchronic administration of Docosahexaenoic acid, investigating functional responses, structural changes proximal and distal to the lesion in primary and secondary injury phases, proteome modulation in injured spinal cord. Docosahexaenoic acid was able i) to restore behavioural responses and ii) to induce pro-regenerative effects and neuroprotective action against demyelination, apoptosis and neuroinflammation. Considering the urgent health challenge represented by spinal injury, this new and reliable mouse model together with the positive effects of docosahexaenoic acid provide important translational implications for promising therapeutic approaches for spinal cord injuries.

Figure 1

Generation of severe spinal cord injury. (A) Representative example of the exposition of the spinal cord in anesthetized animal mounted on a stereotaxic apparatus. (B) Spinal adaptors are connected to a cortical PinPoint precision impactor device (Stoelting), being the impactor positioned on T9-T11 vertebrae. The graphical impact parameters are used to identify potential outliers. To obtain a severe spinal trauma the following parameters are set: - middle, round and flat tip (#4); - velocity 3 m/sec; - depth 5 mm; - dwell time 800 ms. (C) Representative example of the impactor on the spinal cord of an anesthetized animal. (D) Representative longitudinal and cross-sectional μCT images of thoracic spinal cord in intact and (E) in SCI mice, magnification of the impact zone at the thoracic level after SCI is shown in the square. (F) Impact generates a perforation of vertebrae bones and some fragments are visible (zoom, arrow). After impact spinal cord appears compressed (bottom).

Figure 2

Behavioural responses, microcystic degeneration and cystic cavitation in the new mouse model of SCI. (A) BMS scores in female mice subjected to different degrees of impact. BMS defines the locomotion in recovery after SCI. The BMS score ranges from 0 to 9, where 0 indicates complete paralysis and 9 normal movement of the hindlimbs. Motor function recovery is significantly different among the three degree of impact (°°p < 0.01 mild vs moderate; ***p < 0.001 mild and moderate vs severe SCI, Tukey/Kramer; n = 5 mice/group). (B) BMS scores and (C) thermal threshold in daily ip saline- (SAL) or DHA-injected (from D1 to D5 after SCI) females. DHA-treated animals (n = 8) show a significant improvement in motor function, as revealed by higher BMS values in comparison with SAL-treated animals (n = 11) (***p < 0.001), and a significant recovery of tail-flick reflex (***p < 0.001) (Tukey/Kramer). (D) Representative images (5X) of small microcysts, as revealed by GFAP (green) immunostaining, 7 days after SCI in SAL- and DHA-treated mice. (E) Representative image (5X) of cavity formation 30 days after SCI in SAL- and DHA-treated mice. (F) Nuclei stained with DAPI in longitudinal representative images (5X) of spinal cord in naïve, SAL- and DHA-treated mice, 60 days after SCI; DHA induced significant (**p < 0.01) beneficial effects, as shown by the percentage increase of regenerated area in the graph (n = 3 mice/group). (G) GFAP (green) staining in longitudinal representative images (5X) of spinal cord in naïve, SAL- and DHA-treated mice 60 days after SCI. The squares represented high magnification images (20x and zoom 2 respectively). The areas 200 μm caudal and cranial in respect to the epicenter cavity are quantified in the graph. Note, in the graph, that DHA significantly reduced GFAP expression at caudal and cranial level (n = 4 sample/group) (*p < 0.05, **p < 0.01 vs SAL Student’s t test).

Figure 3

Morphometric analysis of astrocytes and microglia after SCI. Representative high magnification (63X, zoom 3X) images of reactive astrocytes (A) and microglia (B) at distal, peri-lesioned and epicenter areas of spinal injured tissues, in saline (SAL) and DHA-treated mice, 7 days (D7) after SCI (n = 3 mice/group). Each image was transformed in digital image, where outline of cell silhouettes were identified and automatically measured for astrocytes, while microglia were singularly counted and divided about the different morphology. (A) Note the increase of GFAP expression and morphological changes showing hypertrophic status of astrocytes, with glial scar formation in the epicenter area, in comparison with naïve non-reactive astrocytes (in the green frame). As shown in graphs, DHA significantly reduced the dimension of astrocytes (quantified by using RGB method that converted pixel in brightness values) in distal and peri-lesioned areas and, even if not statistically significant, in the epicenter, where also phenotypic changes occurred in comparison with SAL-injected mice. (B) After SCI microglia show a hyperactive condition characterized by several phenotypes, differently distributed in the analysed areas: from resting (R) to hypertrophic/bushy (H/B) and un-ramified/amoeboid (U/A) states. As evidenced in graphs, DHA significantly increased the R state in all areas investigated. The increase of the H/B phenotype observed at the epicenter supports phagocytic activity, which may contribute to homeostasis reinstatement. **p < 0.01, ***p < 0.001 vs SAL Student’s t test.

Figure 4

Myelination after SCI: Expression of Myelin Basic Protein. (A) Representative images (63X, zoom 2) of MBP expression in naïve and saline (SAL)- and DHA-treated mice at the epicenter (EPI) and (B) peri-lesioned (PERI) areas, 7 (D7) and 30 days (D30) after SCI. The quantification of MBP, in graph, showed significant increase of MBP expression in SAL EPI at D7 and SAL PERI at D30 in comparison with naïve, while DHA significantly differed from SAL (n = 3 mice/group) (°°°p < 0.001 vs naïve; ***p < 0.001 vs SAL; ^§§p < 0.01 SAL PERI vs SAL EPI. Tukey/Kramer). (C) Western blot analysis and quantification of MBP in naïve/N, saline (S)(SAL) and DHA-treated mice 4 hours (4 H), 24 hours (24 H) and 7 days (D7) after SCI. The time dependent increase of MBP in injured tissues of SAL mice was gradually reduced in DHA-treated animals, where the expression of MBP reached the naïve values at D7. At D30 (graph on the right) MBP expression in DHA is significantly reduced compared to SAL-treated samples (n = 3 mice/group/time point). (°p < 0.05, °°p < 0.01, °°°p < 0.001 vs naïve; *p < 0.05, ***p < 0.001 vs SAL; Dunn’s Test).

Figure 5

Apoptosis in primary injury phase. Representative examples of high magnification (63X, zoom 2) confocal images of Neurons (NeuN, A and B) or oligodendrocytes (OLIG1, C and D) (green), Caspase-3 (red) and their colabeling in ventral horns (VH) of injured spinal cord from saline- (SAL) or DHA-treated animals, 7 days after SCI, at the epicenter (EPI) and peri-lesioned (PERI) areas. DAPI was used to stain nuclei. Manders’ coefficient was utilized to evaluate co-staining between NeuN+ or OLIG-1+ cells and Caspase-3: the R value approximating +1 or −1 indicates linear correlation and 0 indicates absence of correlation. (E) Representative images of NeuN positive cells (green) and quantification of neurons (n = 3 mice/group) in ventral horn (VH NeuN+ cells) 30 days after SCI: DHA significantly (p < 0.05; Student’s t-test) counteracted neuronal loss. (F) Brightness values of Caspase-3 expression at the epicenter and perilesioned areas in ventral horns of spinal cord from naïve, SAL- and DHA-treated mice (n = 3 mice/group). In comparison with naïve animals, SCI induced a higher expression of Caspase-3 at the epicenter, significantly reduced by DHA treatment. After SCI Caspase-3 expression was less enhanced in the perilesioned area of SAL-treated animals, while a higher expression was present in DHA-treated mice. (G) Brightness values of OLIG-1 expression was significantly enhanced after SCI in ventral horns of spinal cord from both saline- and DHA-treated mice, mainly at the epicenter but also in perilesioned area, in comparison with naïve (n = 3 mice/group). °°p < 0.001, °°°p < 0.0001 vs naïve; **p < 0.001 vs SAL. Tukey/Kramer).