Poor Corticospinal Motor Neuron Health Is Associated with Increased Symptom Severity in the Acute Phase Following Repetitive Mild TBI and Predicts Early ALS Onset in Genetically Predisposed Rodents

Abstract

Traumatic brain injury (TBI) is a well-established risk factor for several neurodegenerative disorders including Alzheimer's disease and Parkinson's disease, however, a link between TBI and amyotrophic lateral sclerosis (ALS) has not been clearly elucidated. Using the SOD1^G93A rat model known to recapitulate the human ALS condition, we found that exposure to mild, repetitive TBI lead ALS rats to experience earlier disease onset and shortened survival relative to their sham counterparts. Importantly, increased severity of early injury symptoms prior to the onset of ALS disease symptoms was linked to poor health of corticospinal motor neurons and predicted worsened outcome later in life. Whereas ALS rats with only mild behavioral injury deficits exhibited no observable changes in corticospinal motor neuron health and did not present with early onset or shortened survival, those with more severe injury-related deficits exhibited alterations in corticospinal motor neuron health and presented with significantly earlier onset and shortened lifespan. While these studies do not imply that TBI causes ALS, we provide experimental evidence that head injury is a risk factor for earlier disease onset in a genetically predisposed ALS population and is associated with poor health of corticospinal motor neurons.

Keywords: amyotrophic lateral sclerosis; chronic traumatic encephalopathy; concussion; corticospinal motor neurons; traumatic brain injury.

Figure 1

Repetitive bilateral mild traumatic brain injury (TBI) results in long term functional deficits in wildtype (WT) and SOD1 rats. (A) Rats in the TBI group received closed-skull bilateral controlled cortical impact (CCI) injuries to the motor cortex once a week for five weeks starting at postnatal day (P) 60 and behavior was monitored using Rotarod and Basso-Beattie-Bresnahan (BBB) scoring. (B) Both WT and SOD1 rats experiencing recurrent TBI (solid black and gray squares, respectively) exhibited significant early rotarod deficits, relative to both WT (dotted black triangle) and SOD1 (dotted gray triangles) sham. While TBI rats, regardless of genotype, appeared to recover following the second injury, after the third injury rotarod scores continued to decline and remained significantly lower than sham for the duration of the study. SOD1 rats in both sham and TBI exhibited disease-related progressive decline starting around week 13. (* SOD1 Sham vs SOD1 TBI, * WT sham vs WT TBI, # SOD1 Sham vs WT Sham, # SOD1 TBI vs WT TBI, Two-way ANOVA p < 0.05).

Figure 2

Repetitive bilateral mild TBI results in long-term mild paralysis in WT rats and earlier disease onset in SOD1 rats. (A) BBB scoring following repetitive mild TBI revealed no signs of paralysis after the first two weeks of injury (red oval). However, paralysis was observed following the 3rd injury and continued to worsen throughout the course of injury (green arrows). WT rats maintained a mild level of paralysis throughout the length of the study. An improvement in BBB score occurred following the final injury, prior to onset of disease in SOD1 rats (blue bracket). (* SOD1 sham SOD1 TBI, * WT sham vs WT TBI, # SOD1 Sham vs WT Sham, # SOD1 TBI vs WT TBI, Two-way ANOVA p < 0.05) (B) Onset of paralysis based on forelimb BBB assessment occurred significantly earlier in SOD1 TBI rats, relative to SOD1 sham rats (155 vs. 171 days, respectively * p < 0.01). (C) Earlier onset of disease did not translate to significantly shortened survival time in SOD1 TBI rats when grouped as a whole, relative to SOD1 sham rats.

Figure 3

Rotarod performance distinguishes between mild and severe motor deficits resulting from repeat mild TBI. (A) TBI rats of each genotype were grouped based on symptom severity at week 6 post-first injury; those with a latency to fall shorter than 50 s on the Rotarod were designated “severe”, while those with a latency to fall longer than 50 s were designated “mild”. (* SOD1 Sham vs SOD1 TBI mild, * WT sham vs WT TBI mild, # SOD1 TBI Severe vs SOD1 Sham and SOD1 TBI mild, # WT TBI Severe vs WT Sham and WT TBI Mild, One-way ANOVA, p < 0.05) (B) When grouped according to symptom severity, both WT and SOD1 animals with mild deficits performed significantly worse than sham animals on the rotarod, while those with severe deficits performed significantly worse than both sham and mild TBI animals of their respective genotype. SOD1 animals exhibited further decline in rotarod performance following disease onset. Deficits were sustained in WT TBI mild and severe animals beyond 21 weeks post-first injury. (* SOD1 Sham vs SOD1 TBI Mild vs SOD1 TBI Severe, * WT sham vs WT TBI Mild vs WT TBI Severe, #SOD1 Sham vs WT Sham, # SOD1 TBI Mild vs WT TBI Mild, Two-way ANOVA p < 0.05).

Figure 4

SOD1 rats with initial severe symptoms following repeat TBI exhibit earlier onset and shortened survival. (A) Forelimb BBB scoring revealed that WT TBI rats with initial severe symptoms maintain a significant, mild level of paralysis throughout the study, relative to both WT sham and mild TBI. When grouped by injury severity, SOD1 rats in the severe group experienced (A,B) an earlier decline in forelimb motor function and (C) shortened survival time relative to SOD1 rats in the sham and mild TBI groups. TBI rats exhibiting only mild initial symptoms did not differ from sham SOD1 rats in terms of disease onset and survival. ((A) * WT sham vs WT TBI Severe, * SOD1 Sham vs SOD1 TBI Severe, # SOD1 vs WT (all groups), Two-way ANOVA p < 0.05. (B,C) Wilcoxon’s signed rank test * p < 0.05).

Figure 5

SOD1 rats with acute severe symptoms following repeat TBI display an early loss of corticospinal motor neurons. Immunostaining for CTIP2 and Neurotrace at (A) low and (B) high magnification showed a significant reduction and apparent disorganization of large CTIP2+ (COUP-TF-interacting protein 2) cells located in layer V of the motor cortex occurring in both WT and SOD1 TBI “severe” rats. Quantitative stereological analysis (C) revealed that this reduction occurred in the acute phase in only rats (both WT and SOD1) that exhibited early severe functional deficits and not those TBI rats that presented with mild phenotypic symptoms (* p < 0.05, error bars signify standard error of the mean, SEM). As expected, SOD1 sham endpoint animals exhibited a disease-related reduction in large CTIP2+ cells (>300 μm) relative to earlier timepoints (p < 0.05) and this was not exacerbated with injury. Importantly, however, SOD1 rats experiencing early severe symptoms following repeat TBI showed an early reduction in large CTIP2+ cells at the acute time point not observed in SOD1 sham or mild rats at this time.