Improved functional outcome after chronic stroke with delayed anti-Nogo-A therapy: A clinically relevant intention-to-treat analysis

Abstract

Many preclinical treatment strategies for stroke have failed when tested in human trials. Although the reasons for these translation failures are multifactorial, one potential concern is the statistical analysis of the preclinical data. One way to rigorously evaluate new therapies is to use an intention-to-treat analysis in preclinical studies. Therefore, in this study, we set out to evaluate the treatment efficacy of a potential clinically relevant therapeutic agent for stroke, i.e., anti-Nogo-A immunotherapy, using an intention-to-treat analysis. Adult rats were trained on the skilled forelimb reaching task and subsequently underwent an ischemic stroke. Nine weeks later, the rats either received intracerebroventricular anti-Nogo-A antibody, control antibody, or no treatment. Skilled reaching performance was assessed by a non-linear model using both an intention-to-treat and per-protocol analysis. Following testing, dendritic complexity was evaluated in the contralesional and perilesional sensorimotor cortex. Both intention-to-treat and per-protocol analysis showed that anti-Nogo-A immunotherapy resulted in statistically significant improved recovery on the skilled forelimb reaching task, although treatment effect was less (though statistically significant) in the intention-to-treat group. Improved functional performance was not shown to be associated with dendritic changes. In conclusion, this study provides evidence for the importance of using intention-to-treat paradigms in testing preclinical therapeutic strategies.

Keywords: Anti-Nogo-A; chronic; recovery; sensorimotor; stroke.

Figure 1.

Experimental design and timeline.

Figure 2.

Experimental overview. Flow chart showing the experimental design and the number of animals at every stage of the study.

Figure 3.

Stroke lesion size and location. (a) Diagram of stroke lesion topology showing the differences in lesion size and location. There was no statistical difference in the stroke size between treatment groups either in the ITT analysis (F(2, 32) = 0.257, p = 0.78). (b) or the PP analysis (F(2,22) = 0.004, p = 0.99). (c). Data presented as mean ± SEM. one-way ANOVA. ITT: Stroke only (n = 10); Stroke/Control Ab (n = 12); Stroke/Anti-Nogo-A Ab (n = 13), PP: Stroke only (n = 8); Stroke/Control Ab (n = 9); Stroke/Anti-Nogo-A Ab (n = 8).

Figure 4.

Functional outcome analysis. Upper panels show weekly raw reaching scores starting from week zero (baseline) to the end of study for ITT (a) and PP (b) analyses. Lower panels show the fitted group-level logistic recovery profiles for each treatment group based on the non-linear model for the ITT (c) and PP (d) analyses. Animals treated with anti-Nogo-A antibody after stroke show significant improvement as compared with animals of the stroke only group in both the ITT (p = 0.01) and PP (p = 0.02) analyses. Animals treated with anti-Nogo-A antibody after stroke show significant improvement as compared with animals of the stroke/control antibody in both the ITT (p = 1.76 e⁻⁶) and PP (p = 2.58 e⁻⁸) analyses. ITT: Stroke Only (n = 10); Stroke/Control Ab (n = 12); Stroke/Anti-Nogo-A Ab (n = 14), PP: Stroke Only (n = 8); Stroke Control Ab (n = 9); Stroke/Anti-Nogo-A Ab (n = 8).

Figure 5.

Dendritic analysis of Layer V pyramidal neurons in contralesional caudal forelimb cortex. Dendritic complexity analyses showed no difference between ITT groups in the numbers of dendritic branches (a) and total dendritic length (b) of apical dendrites, as well as in the numbers of dendritic branches (c) and total dendritic length (d) of basilar dendrites in the contralesional caudal forelimb cortex. Data presented as mean ± SEM. p > 0.05, one-way ANOVA. Stroke Only (n = 10); Stroke/Control Ab (n = 7); Stroke/Anti-Nogo-A Ab (n = 13).

Figure 6.

Dendritic analysis of Layer V pyramidal neurons in perilesional caudal forelimb cortex. Dendritic complexity analyses showed no difference between ITT groups in the numbers of dendritic branches (a) and total dendritic length (b) of apical dendrites, as well as in the numbers of dendritic branches (c) and total dendritic length (d) of basilar dendrites in the perilesional caudal forelimb cortex. Data presented as mean ± SEM. p > 0.05, one-way ANOVA. ITT: Stroke Only (n = 6); Stroke/Control Ab (n = 6); Stroke/Anti-Nogo-A Ab (n = 8).