Preventing flow-metabolism uncoupling acutely reduces axonal injury after traumatic brain injury

Abstract

We have previously presented evidence that the development of secondary traumatic axonal injury is related to the degree of local cerebral blood flow (LCBF) and flow-metabolism uncoupling. We have now tested the hypothesis that augmenting LCBF in the acute stages after brain injury prevents further axonal injury. Data were acquired from rats with or without acetazolamide (ACZ) that was administered immediately following controlled cortical impact injury to increase cortical LCBF. Local cerebral metabolic rate for glucose (LCMRglc) and LCBF measurements were obtained 3 h post-trauma in the same rat via ¹⁸F-fluorodeoxyglucose and ¹⁴C-iodoantipyrine co-registered autoradiographic images, and compared to the density of damaged axonal profiles in adjacent sections, and in additional groups at 24 h used to assess different populations of injured axons stereologically. ACZ treatment significantly and globally elevated LCBF twofold above untreated-injured rats at 3 h (p<0.05), but did not significantly affect LCMRglc. As a result, ipsilateral LCMRglc:LCBF ratios were reduced by twofold to sham-control levels, and the density of β-APP-stained axons at 24 h was significantly reduced in most brain regions compared to the untreated-injured group (p<0.01). Furthermore, early LCBF augmentation prevented the injury-associated increase in the number of stained axons from 3-24 h. Additional robust stereological analysis of impaired axonal transport and neurofilament compaction in the corpus callosum and cingulum underlying the injury core confirmed the amelioration of β-APP axon density, and showed a trend, but no significant effect, on RMO14-positive axons. These data underline the importance of maintaining flow-metabolism coupling immediately after injury in order to prevent further axonal injury, in at least one population of injured axons.

FIG. 1.

A brain atlas coronal section (Paxinos and Watson, 1997) illustrating the core contused region (hatched area) at −2.80 mm to the bregma. Measurements for LCMRglc and LCBF and the density of β-APP-immunoreactive profiles were determined in the following regions of interest: (1) contusion core, (2) pericontusion margin, (3) cingulum, (4) corpus callosum, (5) external capsule, (6) internal capsule, (7) CA1, (8) CA3, (9) dentate gyrus, and (10) dorsolateral thalamus (LCBF, local cerebral blood flow; LCMRglc, local cerebral metabolic rate for glucose; β-APP, β-amyloid precursor protein).

FIG. 2.

Laser Doppler flow measurements of local cerebral blood flow (LCBF) over the contralateral parietal cortex immediately after injury, before, and after injection of acetazolamide (100 mg/kg⁻¹, open symbols; and 150 mg/kg⁻¹, closed symbols). Data are expressed relative to pre-injection levels. Values are means±standard error; n=3 per group). Difference between the groups was significant (p<0.05).

FIG. 3.

Representative local cerebral blood flow (LCBF) parametric maps from an injured untreated (A) and an acetazolamide (ACZ)-treated rat (B) at 3 h after injury, and (C) bar graphs of quantified contralateral and ipsilateral LCBF values (as percentages of the corresponding sham control region of interest [ROI] values) in untreated-injured (open bars), and ACZ-treated rats (solid bars), at 3 h post-trauma in grey and white matter (shaded area) brain regions. Administration of ACZ resulted in a global increase in LCBF that was maintained in both grey and white matter regions until 3 h post-injury (A versus B). LCBF values were significantly increased bilaterally in most regions of the brain compared to untreated, injured rats, such that values were similar to or above those in sham-injured rats (horizontal line). Data are plotted as means±standard error of the mean; *p<0.05, **p<0.01 corrected for multiple comparisons; Core, contusion core; Marg, pericontusion margin; Cing, cingulum; Thal, thalamus; CC, corpus callosum; EC, external capsule; IC, internal capsule; DG, dentate gyrus). Color image is available online at www.liebertonline.com/neu

FIG. 4.

Representative local cerebral metabolic rate for glucose (LCMRglc) parametric maps from an injured, untreated (A) and acetazolamide (ACZ)-treated rat (B) at 3 h after injury, and (C) bar graphs of quantified contralateral and ipsilateral LCMRglc values (as percentages of the corresponding sham control region-of-interest [ROI] values) in untreated-injured (open bars) and ACZ-treated rats (solid bars) at 3 h post-trauma in grey and white matter (shaded area) brain regions. Despite the observation that injury-induced hyperglycolysis (the high LCMRglc values in A) was often normalized by ACZ (A versus B), wide variability between animals (e.g., ipsilateral and marginal zone, C) resulted in the finding that there was no significant effect of ACZ treatment on LCMRglc in any brain region measured. Data are plotted as means±standard error of the mean; the horizontal line represents sham-injured values (Core, contusion core; Marg, pericontusion margin; Cing, cingulum; Thal, thalamus; CC, corpus callosum; EC, external capsule; IC, internal capsule; DG, dentate gyrus). Color image is available online at www.liebertonline.com/neu

FIG. 5.

Representative parametric maps from an injured, untreated (A) and acetazolamide (ACZ)-treated rat (B) at 3 h after trauma for the calculated ratio of LCMRglc: LCBF. The ratio between flow and metabolism was decreased globally by ACZ (B), and this extended to the pericontusion margin (arrows). (C) Bar graph demonstrating ipsilateral LCMRglc:LCBF ratio values (as percentages of the corresponding sham-control region-of-interest [ROI] data) in untreated-injured (open bars) and ACZ-treated rats (solid bars) in grey and white matter (shaded area) brain regions. ACZ significantly reduced the ratios in most regions of the ipsilateral hemisphere, including the pericontusion margin. Data are plotted as means±standard error of the mean; the horizontal line represents sham-injured values. (Core, contusion core; Marg, pericontusion margin; Cing, cingulum; Thal, thalamus; CC, corpus callosum; EC, external capsule; IC, internal capsule; DG, dentate gyrus; LCMRglc:LCBF, local cerebral metabolic rate for glucose:local cerebral blood flow ratio). Color image is available online at www.liebertonline.com/neu

FIG. 6.

(A) High-power views showing β-APP immunoreactivity in different ipsilateral brain regions at 1 day after injury in ACZ-treated animals compared with sham-control and untreated-injured animals. Note that the number of damaged immunoreactive axons (arrows) was significantly reduced in ACZ-treated animals compared with untreated-injured animals (scale bar=20 μm). (B) Bar graph of the mean densities of immunopositive profiles displaying β-APP immunoreactivity in ACZ-treated (solid bars) and untreated-injured rats (open bars) at 3 and 24 h after trauma. There was a significant decrease in the number of β-APP-immunopositive damaged axons in the cingulum, corpus callosum, pericontusion margin, and thalamus in the ACZ group at 24 h. Data are plotted as means±standard error of the mean (Marg, pericontusion margin; Cing, cingulum; Thal, thalamus; CC, corpus callosum; EC, external capsule; IC, internal capsule; ACZ, acetazolamide; β-APP, β-amyloid precursor protein).

FIG. 7.

(A) Representative coronal montages of β-APP immunostaining within the corpus callosum and cingulum under the impact zone ipsilaterally at 24 h in a vehicle-treated rat demonstrating the contoured region in which the stereological analyses were performed. (A`) A montage image of the corresponding contralateral hemisphere montage of the same vehicle-injured rat showing an absence of β-APP immunostaining in the white matter (contoured area). (B) Representative high-power images showing positive staining for β-APP (green), and RMO14 (red) in the corpus callosum of a vehicle-treated, injured rat at 24 h. Consistent with previously reported results, there is no overlap between staining, which suggests that impaired axonal transport and neurofilament compaction may be occurring in either different populations of axons, or in different parts of the same axon. (C and D) Representative high-power confocal z-stack images of the corpus callosum underlying the injury core immunostained for β-APP (green) and RMO14 (red) from (C) a vehicle-treated injured rat, and (D) an ACZ-treated injured rat. Bulb-like swelling and punctate profiles (arrowheads) are associated with β-APP-positive axons. RMO14-positive axons are thin and elongated or display vacuolization (arrows). Note the overall decrease in the amount of positive staining for both β-APP and RMO14 in ACZ compared to vehicle animals (scale bars=10 μm). (D`) A representative β-APP/RMO14 image of the contralateral corpus callosum of a vehicle-injured rat acquired with the same acquisition parameters (gain/offset) as the other high-powered images. The absence of RMO14-positive profiles in this region indicates that axonal injury is specific to the ipsilateral callosum at this post-injury time point. Bar graphs of the stereologically-estimated number of ipsilateral (E) β-APP, and (F) RMO14-positive axons in saline vehicle-treated (SAL, open bars), and ACZ-treated (ACZ, solid bars) at 24 h post-injury as determined by stereology. Data are plotted as means±standard error of the mean, and individual data from each rat are over-plotted (closed dots; **p<0.01; ACZ, acetazolamide; β-APP, β-amyloid precursor protein). Color image is available online at www.liebertonline.com/neu