Fig 1.
Enhanced gelatinases detected at 48 h after TBI.
(A) Gelatin zymography from representative brain lysates indicates increased expression of MMP-9 and MMP-2 at 48 h post-injury as compared to sham controls. The pro-forms of MMP-9 and MMP-2 were detected at ~105 and 72 kDa, respectively. Purified human MMP-2 and MMP-9 were used as standards. (B) Quantification of band intensity revealed a robust increase in the pro-enzyme forms of MMP-2 and (C) MMP-9, as well as increases in the active enzyme forms after TBI compared to sham (n = 6 per group; unpaired t-tests, *p<0.05, **p<0.01 and ***p<0.001, TBI compared to sham). Bars represent mean + sem.
Fig 2.
Early gelatinase inhibition does not impact injury-induced hyperactivity, measures of anxiety, or motor function at adulthood after pediatric TBI.
(A) Injury resulted in hyperactivity by adulthood, indicated by increased total distance traveled in an open field (2-way ANOVA, effect of injury, **p<0.01). (B) The percent time spent in center of the open field, a measure of anxiety, was not altered by either injury or treatment (2-way ANOVA n.s.). (C) Anxiety was also measured by percent time spent in the open arms of the elevated plus maze, and neither injury nor p-OH SB-3CT treatment affected this measure (2-way ANOVA n.s.). (D) Motor function, evaluated by latency to fall from an accelerating rotarod, was similar in all groups across consecutive testing days (2-way RM ANOVA). Bars represent mean + sem and values represent mean ± sem (n = 15/group).
Fig 3.
Cognitive deficits detected in the Morris water maze (MWM) at adulthood after pediatric TBI, are unaffected by gelatinase inhibition.
(A) During the visible sessions, quantification of latency to reach the platform revealed an impairment in task learning by TBI mice compared to sham controls (multivariate ANOVA overall effect of TBI, **p<0.01). (B) During hidden platform sessions, injured mice also showed a greater latency to reach the platform as compared to sham controls (overall effect of TBI, ***p<0.001), indicating an impairment in spatial memory. Cumulative distance to the target was also quantified as an alternative outcome measure (C-D), which similarly detected impairments in task performance and spatial memory in TBI mice compared to sham controls (overall effect of TBI, **p<0.01). (E) Probe trial performance was quantified as cumulative distance to the target. Injured mice traveled a greater distance to reach the target quadrant compared to sham controls (RM ANOVA, overall effect of TBI, **p<0.01) (n = 15/group). Bars represent mean + sem and values represent mean ± sem.
Fig 4.
Acute gelatinase inhibitor does not attenuate deficits in social behavior at adulthood after pediatric TBI.
(A) Social investigation was quantified by the resident-intruder paradigm, revealing that TBI mice as compared to sham controls spent less time investigating a naïve intruder mouse (2-way ANOVA overall effect of TBI, **p<0.01). (B) In the three-chamber social approach task (stage 2), all mice showed an overall preference for sociability with stimulus mouse 1 compared to the empty chamber (2-way RM ANOVA overall effect of chamber, p = 0.0003). (C) Stage 3 of the three-chamber task tested social novelty. Here, sham-operated mice revealed a preference for a novel stimulus mouse compared to the now-familiar mouse (2-way RM ANOVA interaction, p = 0.0055; subsequent Sidak’s post-hoc tests, ***p<0.001, ****p<0.0001 as indicated graphically). In contrast, TBI mice showed a lack of of social memory (n.s. by Sidak's post-hoc) (n = 15/group). Bars represent mean + sem.
Fig 5.
Gelatinase inhibition with p-OH SB-3CT does not attenuate extensive injury-induced loss of cortical and hippocampal structures.
Volumetric estimates spanning Bregma 1.5 to -3.8mm in the cortex (Ctx; A), hippocampus (Hpc; B) and dentate gyrus (DG; C) revealed injury-induced reductions (unpaired t-tests *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 as indicated graphically; n = 11–15/group). (D) Unbiased cell counts performed in the ipsilateral DG found similar numbers of surviving neurons in the upper and lower blades of injured mice independent of drug treatment (n = 8–9/group). Bars represent mean + sem.
Fig 6.
p-OH SB-3CT achieved therapeutic concentrations within the immature brain and attenuates gelatinase activity.
A) Concentrations are depicted as μM for plasma and pmol/mg tissue for brain (equivalent to μM assuming a density of 1g/mL). Levels of p-OH SB-3CT peaked at 30 min after the final dose, in both plasma and brain, and declined by 4 h (n = 3 per time point). Values are mean ± sd. B) Representative images of in-situ zymography of brain sections with fluorogenic substrate DQ-Gel (green in top panels) and merged with nuclear staining with DAPI (blue in bottom panels), scale bar is 50 μm.
Table 1.
Concentrations of p-OH SB-3CT after multiple-dose s.c. administration.