Anatomical and behavioral characterization of three hemiplegic animal models

Abstract

Background: Hemiplegia is characterized by muscle weakness on one side of the body, often resulting from damage to the brain, spinal cord, or associated nerves. This condition commonly occurs due to strokes, traumatic brain injuries (TBI), or spinal cord injuries (SCI), which can damage corticospinal neurons (CSNs) and the corticospinal tract (CST). However, there is still a notable lack of comprehensive studies that systematically characterize the anatomical and behavioral aspects of these hemiplegic animal models.

Objective: This study aimed to validate and compare existing models of TBI, stroke, and SCI in order to identify the most suitable preclinical hemiplegia models for future research.

Method: Using viral-based retrograde tracing, we first mapped the cortical distribution of CSNs responsible for hindlimb movement. Anterograde and retrograde viral tracing techniques were then employed to label and evaluate the damage to CSNs and the CST in three models: photothrombotic stroke, Feeney's weight-drop TBI, and T10 hemi-section SCI. We also conducted behavioral tests to assess spontaneous motor function recovery, including open field and rotarod tests for gross motor function, as well as beam walking and irregular ladder walking tasks for assessing skilled motor function.

Results: Our findings revealed that the CSNs controlling hindlimb movement are concentrated in the hindlimb region of the primary somatosensory cortex (S1HL). In the TBI and stroke models, there was complete destruction of ipsilateral CSNs in the S1HL and loss of CST fibers governing hindlimb movement. In the SCI model, ipsilateral CST fibers below T10 were also lost. After 8 weeks post-injury, all three groups of hemiplegic mice showed improvements in motor function, with gross motor function returning to normal levels; however, the recovery of skilled motor function was only modest. Notably, the degree of improvement in fine motor skills varied among the hemiplegia models, with mice subjected to brain injury (stroke and TBI) demonstrating significantly greater recovery in fine motor skills compared to those with SCI.

Conclusion: We confirmed and validated previous hemiplegia models by damaging CSNs or CST controlling hindlimb movement. Post-injury, gross motor function gradually returned to normal levels across all groups, whereas recovery of skilled motor function was limited. Furthermore, there were significant differences in the recovery of skilled motor function between brain injury models and the SCI model. These hemiplegic mouse models are valuable tools for studying post-injury skilled motor functions.

Clinical trial number: Not applicable.

Keywords: Hemiplegia; Motor function recovery; Spinal cord injury; Stroke; Traumatic brain injury.

Fig. 1

Stable stroke mice model. A TTC staining of mice brain at 24 h after stroke. Scale bar:1 cm. B The expression of GFP in the cerebral cortex and brainstem was examined after the lumbar spinal cord injection of the retrograde tracing virus AAV/Retro-GFP in the mice of the sham group and the stroke group. Scale bars: 1 mm in cortex and 100 μm in brainstem. C Quantification of corticospinal neurons (CSNs) in contralateral and ipsilateral corticesin sham and stroke groups. D Quantification of mean fluorescence intensity (MFl) of corticospinal tract (CST) in contralateral and ipsilateral brainstem in sham and stroke groups. sham, n = 5 mice; stroke, n = 5 mice. Student’s t test, ***p < 0.001

Fig. 2

Stable traumatic brain injury (TBI) model. A Nissl staining of mice brain sections at 1 week after TBI. Scale bar: 1 mm. B The expression of GFP positive CSNs and CST in the cerebral cortex and brainstem was examined after the lumbar spinal cord injection of the retrograde tracing virus AAV/Retro-GFP in the mice of the sham group and the TBI group. Scale bars: 1 mm in cortex and 100 μm in brainstem. C Quantification of contralateral and ipsilateral CSNs in sham and TBI groups. D Quantification of MFI of contralateral and ipsilateral CST in sham and TBI groups. sham, n = 5 mice; TBI, n = 5 mice. Student’s t test, ***p < 0.001

Fig. 3

Stable spinal cord injury (SCI) model. (A) Diagram of the hemi-section SCI model and the outcome of GFAP staining. The lateral hemi-section at the T10 leads to interrupting of ipsilateral CST projection. Immunofluorescent staining for anti-GFAP in the spinal cord of the sham and T10 SCI mice. Scale bar: 500 μm. (B) Diagram of the virus injection and pictures of L5 spinal cord sections. AAV-ChR2-mCherry was injected into the contralesional sensorimotor cortex to label CST. mCherry positive CST fibers images in sham and T10 SCI mice were displayed. (C) The MFI of mCherry fibers in L5 of sham and SCI groups. sham, n = 5 mice; SCI, n = 5 mice. Student’s t-test, ***p < 0.001

Fig. 4

Fine motor function tests of three types of hemiplegia mice. A Diagram of beam walking. B The error rates on the beam walking during the spontaneous recovery from the baseline to the week 8 after injury in three groups of hemiplegia mice and the sham group. Two-way Repeated Measure ANOVA. (C–E) The percentage of recovery rate on the beam walking between baseline, 1 day and week 8 after injury in three groups of hemiplegia mice. sham, n = 6 mice; stroke, n = 6 mice; TBI, n = 6 mice; SCI, n = 6 mice. One-way ANOVA. F Diagram of irregular ladder walking test. G The error rates on the irregular ladder walking during the spontaneous recovery from baseline and week 8 after injury in three groups of hemiplegia mice and the sham group. Two-way Repeated Measure ANOVA. H–J The percentage of recovery rate on the irregular ladder walking between baseline, 1 day and the week 8 after injury in three groups of hemiplegia mice. sham, n = 6 mice; stroke, n = 6 mice; TBI, n = 6 mice; SCI, n = 6 mice. One-way ANOVA. ^@@@p < 0.001, ^@@p < 0.01, and ^@p < 0.05 for sham vs. stroke; ^&&&p < 0.001, ^&& p < 0.01, and ^&p < 0.05 for sham vs. TBI; ^###p < 0.001, ^##p < 0.01, and ^#p < 0.05 for sham vs. SCI; ***p < 0.001, **p < 0.01, ^※※※p < 0.001, ^※※p < 0.01, and ns = not significant

Fig. 5

Gross motor function tests of three types of hemiplegia mice. A Diagrams of rotarod test. B The latency to fall from the rotarod during spontaneous recovery from baseline to the week 8 after injury in the three groups of hemiplegia mice and the sham group. Two-way Repeated Measure ANOVA; C–E The percentage of recovery rate in the rotarod test between baseline to the 1 day and week 8 after injury in the three hemiplegia groups. stroke, n = 6 mice; TBI, n = 6 mice; SCI, n = 6 mice. One-way ANOVA; F Diagrams of open-field test. G The total distances in the open field during spontaneous recovery from baseline to the week 8 after injury in the three groups of hemiplegia and the sham group. Two-way Repeated Measure ANOVA. H–J The percentage of recovery rate in the open field between baseline to the 1 day and week 8 after injury in the three hemiplegia groups. sham, n = 5 mice; stroke, n = 5 mice; TBI, n = 5 mice; SCI, n = 5 mice. One-way ANOVA. ^@@@p < 0.001, ^@@p < 0.01, and ^@p < 0.05 for sham vs. stroke; ^&&&p < 0.001, ^&& p < 0.01, and ^&p < 0.05 for sham vs. TBI; ^###p < 0.001, ^##p < 0.01, and ^#p < 0.05 for sham vs. SCI; ***p < 0.001, **p < 0.01, *p < 0.05, and ns = not significant