Differential effects of FK506 on structural and functional axonal deficits after diffuse brain injury in the immature rat

Abstract

Diffuse axonal injury is a major component of traumatic brain injury in children and correlates with long-term cognitive impairment. Traumatic brain injury in adult rodents has been linked to a decrease in compound action potential (CAP) in the corpus callosum, but information on trauma-associated diffuse axonal injury in immature rodents is limited. We investigated the effects of closed head injury on CAP in the corpus callosum of 17-day-old rats. The injury resulted in CAP deficits of both myelinated and unmyelinated fibers in the corpus callosum between 1 and 14 days postinjury (dpi). These deficits were accompanied by intra-axonal dephosphorylation of the 200-kDa neurofilament subunit (NF200) at 1 and 3 dpi, a decrease in total NF200 at 3 dpi and axonal degeneration at 3 and 7 dpi. Although total phosphatase activity decreased at 1 dpi, calcineurin activity was unchanged. The calcineurin inhibitor, FK506, significantly attenuated the injury-induced NF200 dephosphorylation of NF200 at 3 dpi and axonal degeneration at 3 and 7 dpi but did not affect the decrease in NF200 protein levels or impaired axonal transport. FK506 had no effect on CAP deficits at 3 dpi but exacerbated the deficit in only the myelinated fibers at 7 dpi. Thus, in contrast to adult animals, FK506 treatment did not improve axonal function in brain-injured immature animals, suggesting that calcineurin may not contribute to impaired axonal function.

Figure 1

Measurement of compound action potential (CAP) in the corpus callosum. (A) Representative schematic of the coronal slice containing the corpus callosum used for recording the CAP. (B) Stimulating and recording electrodes were placed approximately 0.5 mm on either side of the midline. (C) Representative trace of a typical CAP used to measure the amplitudes. The amplitude of the N1 component (myelinated fibers) was the difference in voltage from the first positive peak to first negative trough; the amplitude of the N2 component (unmyelinated fibers) was measured by dropping a tangent from the baseline to the second negative trough. The duration of N1 was measured as the time between the first and second positive peaks; the duration of N2 was measured as the time between the second positive peak until the signal returned to baseline.

Figure 2

Effect of diffuse brain injury on the amplitude of the compound action potentials of axons in the corpus callosum. (A) Representative traces from slices obtained from sham- and brain-injured at 1, 7 and 14 days following surgery/injury. (B-E) Input-output curves of amplitude for the N1 and N2 components at 1 (B, D), 7 and 14 days (C, E). Although the graphs are separated by time, statistical analyses were performed by combining all time points.

Figure 3

Effect of diffuse brain injury on the electrophysiologic properties of the axons in the corpus callosum. (A-F) Graphs representing duration (A, B), conduction velocity (C, D) and refractoriness (E, F) of the N1 (A, C, E) and N2 components (B, D, F). Filled (sham-injured) and open (brain-injured) bars represent mean values; error bars represent SDs. All p values are significant compared to their respective sham-injured animals. *, p < 0.05; **, p < 0.005; #, p < 0.01; ##, p < 0.001; ###, p < 0.0001.

Figure 4

Intra-axonal accumulation of dephosphorylated 200-kDa neurofilament subunit following diffuse brain injury in the immature rat. (A-F) Representative photomicrographs of SMI-32-labeled axons within the corpus callosum of sham (A), and brain-injured rats at 1 (B) and 3 days (C) post-injury. (D) An example of SMI-32-labeled swollen contiguous axons. (E, F) Examples of terminal bulbs. (G-I) An example of double-label immunofluorescence for intra-axonal amyloid precursor protein (APP) accumulation (red) and SMI-32 immunoreactivity (green) at 1 day post-injury. Single-labeled profiles (either APP-positive/SMI-32-negative or APP-negative/SMI-32-positive) are denoted by arrows; double-labeled profiles (APP-positive/SMI-32-positive) are denoted by arrowheads. Photomicrographs were obtained at 63x magnification. Scale bars in panels C (20x) and F (100x) represent 100 μm for panels A-C and D-F, respectively.

Figure 5

Alterations in total 200-kDa neurofilament subunit (NF200) following diffuse brain injury in the immature rat. (A-C) Representative photomicrographs of NF200-labeled axons from a sham-injured rat (A), and injured rats at 1 (B) and 3 days (C) post-injury. Arrows denote examples of NF200-labeled axonal accumulation. (D) Representative immunoblots of NF200 and actin (loading control) using lysates of the subcortical white matter. (E) Quantification of the optical density of NF200 relative to that of actin. *, p < 0.005. Scale bar = 100 μm for all panels.

Figure 6

Phosphatase activity following diffuse brain injury in the immature rat. (A, B) Total and EGTA-sensitive (calcineurin) phosphatase activities were measured using lysates of the subcortical white matter (A) or cortex (B). Shaded bars represent average calcineurin activity; the unfilled part represents average EGTA-insensitive activity. Error bars = SD. *, p < 0.05.

Figure 7

Effect of FK506 on dephosphorylation and expression of 200-kDa-neurofilament subunit following diffuse brain injury in the immature rat. (A-D) Representative SMI-32 photomicrographs of vehicle-treated animals at 1 (A) and 3 (B) days post-injury, and FK506-treated animals at 1 (C) and 3 (D) days post-injury. E: Quantification of SMI-32-positive profiles in subcortical white matter tracts using the grid method. IHC = immunohistochemistry. (F, G) Representative NF200 photomicrographs of vehicle- (F) and FK506-treated (G) at 3 days post-injury. (H) Quantification of NF200-positive profiles in subcortical white matter tracts. (I) Representative immunoblots of NF200 and actin (loading control) of lysates from subcortical white matter tracts at 3 days post-injury. (J) Quantification of optical density of NF200 relative to that of actin. The increase in the NF200 expression in the FK506-treated injured animals was not significant. *, p < 0.05; ##, p < 0.001. Scale bar = 100 μm for all panels.

Figure 8

Effect of FK506 on impaired axonal structure and function following diffuse brain injury in the immature rat. (A-D) Representative micrographs of amyloid precursor protein (APP) immunoreactivity within the corpus callosum of vehicle-treated animals at 1 (A) and 3 (C) days post-injury and FK506-treated animals at 1 (B) and 3 (D) days post-injury. (E) Quantification of the area of APP immunoreactivity. (F-I) Representative micrographs of Fluoro-Jade B (FJB) reactivity in the corpus callosum of vehicle-treated animals at 3 (F) and 7 (H) days post-injury and FK506-treated animals at 3 (G) and 7 (I) d post-injury. (J) Quantification of the density of FJB-positive profiles in the corpus callosum. Panels K and L illustrate the effect of vehicle or FK506 administration on the amplitude of myelinated and unmyelinated fibers, respectively, within the corpus callosum at 3 and 7 days post-injury. *, p < 0.05, ##, p < 0.001, ###, p < 0.0001. Scale bar = 100 μm for all panels.