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Minocycline inhibits contusion-triggered mitochondrial cytochrome c release and mitigates functional deficits after spinal cord injury - PubMed

  • ️Thu Jan 01 2004

Minocycline inhibits contusion-triggered mitochondrial cytochrome c release and mitigates functional deficits after spinal cord injury

Yang D Teng et al. Proc Natl Acad Sci U S A. 2004.

Abstract

We investigated whether permeability transition-mediated release of mitochondrial cytochrome c is a potential therapeutic target for treating acute spinal cord injury (SCI). Based on previous reports, minocycline, a second-generation tetracycline, exerts neuroprotection partially by inhibiting mitochondrial cytochrome c release and reactive microgliosis. We first evaluated cytochrome c release at the injury epicenter after a T10 contusive SCI in rats. Cytochrome c release peaked at approximately 4-8 h postinjury. A dose-response study generated a safe pharmacological regimen that enabled i.p. minocycline to significantly lower cytosolic cytochrome c at the epicenter 4 h after SCI. In the long-term study, i.p. minocycline (90 mg/kg administered 1 h after SCI followed by 45 mg/kg administered every 12 h for 5 days) markedly enhanced long-term hind limb locomotion relative to that of controls. Coordinated motor function and hind limb reflex recoveries also were improved significantly. Histopathology suggested that minocycline treatment alleviated later-phase tissue loss, with significant sparing of white matter and ventral horn motoneurons at levels adjacent to the epicenter. Furthermore, glial fibrillary acidic protein and 2',3' cyclic nucleotide 3' phosphodiesterase immunocytochemistry showed an evident reduction in astrogliosis and enhanced survival of oligodendrocytes. Therefore, release of mitochondrial cytochrome c is an important secondary injury mechanism in SCI. Drugs with multifaceted effects in antagonizing this process and microgliosis may protect a proportion of spinal cord tissue that is clinically significant for functional recovery. Minocycline, with its proven clinical safety, capability to cross the blood-brain barrier, and demonstrated efficacy during a clinically relevant therapeutic window, may become an effective therapy for acute SCI.

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Figures

Fig. 4.
Fig. 4.

(a and b) GFAP immunocytochemistry demonstrating reduced astrogliosis in rat spinal cord white matter receiving minocycline (a) vs. equivolumetric saline vehicle (b). (Scale bar = 250 μm.) (c) Hypertrophic astrocyte of a rat receiving saline vehicle treatment. (Scale bar = 20 μm.) (d) The average number of reactive astrocytes (y axis, number of astroglia per 20 μm of tissue) was significantly lower in the treated group (hatched bar) vs. the controls (open bar) (n = 8 rats per group; P = 0.017, unpaired Student's t test).

Fig. 1.
Fig. 1.

(a and b) Cytochrome c Western blots of cytosolic (a) and mitochondrial (b) fractions of precontusion and postcontusion spinal cord tissue at the epicenter. (c) Dose-dependent effect of minocycline on cytochrome c release at the injury epicenter, with significant blocking at the 90 and 180 mg/kg doses. COX IV, cyclooxygenase IV.

Fig. 2.
Fig. 2.

Effect of minocycline on the recovery of coordinated hind limb functions. (a) Comparison of groups (n = 10 rats per group) that received vehicle alone or minocycline in inclined plane performance. Data points represent the average ± SEM maximum angle at which rats can maintain position for 5 s. Data were analyzed with repeated-measures ANOVA, which showed an overall significant (P < 0.05) effect of treatment. Asterisks indicate that means are significantly different from the control group at the specified times after SCI (Student's t test). When facing upward, animals in the two groups (▴ and ▵) were statistically similar for all time points, indicating that they were similar in general body condition. When facing downward, the minocycline-treated group (▾) performed significantly better starting at day 7 p.i., compared with the vehicle control group (▿). (b) Locomotor function graded on the BBB scale that ranges from 21 in normal rats to 0 in rats with complete hind limb paralysis (25). Data points represent the group average ± SEM. Results were analyzed with repeated-measures ANOVA, which showed an overall significant (P < 0.05) effect of treatment. Asterisks indicate that means are significantly different from the vehicle-treated control group at the specified times after SCI (Student's t test).

Fig. 3.
Fig. 3.

(a) Lack of significant effect of minocycline (total dose of 495 mg/kg per rat over 5 days) on lesion volume at the injury epicenter and adjacent tissue up to 4 mm rostral and caudal to the epicenter, in 1-mm increments (solvent blue and hematoxylin/eosin staining). (b) Representative transverse spinal cord sections after T10 contusion injury at the epicenter and in 1-mm increments rostral and caudal to the epicenter in a rat receiving minocycline (Upper) and saline vehicle (Lower). Epicenter sections are marked by arrows. (c) Overall significant difference (P = 0.014, repeated-measures ANOVA) between rats receiving minocycline or saline vehicle (n = 10 rats per group) in the average area of residual total white matter (i.e., white matter and hypomyelinated white matter; solvent blue and hematoxylin/eosin staining) (24), as well as 2, 3, and 4 mm rostral and 3 and 4 mm caudal to the lesion epicenter (P < 0.03, unpaired Student's t test). (d) Minocycline treatment significantly protected ventral horn neurons 3 and 4 mm rostral to the epicenter (P < 0.03, repeated-measures ANOVA), as well as 2, 3, and 4 mm caudal to the epicenter (P < 0.05, unpaired Student's t tests). (e and f) Representative transverse cresyl violet-stained sections 2 mm caudal to the injury epicenter in a rat receiving minocycline (e) or saline vehicle (f). (Scale bar = 50 μm.)

Fig. 5.
Fig. 5.

(a and b) CNPase immunocytochemistry demonstrating increased oligodendrocyte preservation in minocycline-treated rat spinal cord ventral funiculi 4 mm caudal to the epicenter (b) vs. that in the controls (a) (n = 8 rats per group). (Scale bar = 75 μm). (c) Anti-CNPase and 4′,6-diamidino-2-phenylindole (DAPI) double-staining, demonstrating oligodendroglia (arrowheads) as well as the nuclei of cells not reacting with the anti-CNPase antibody (arrows). (d) Composite histograms of total luminosities summarized from each experimental group showed a significant difference in anti-CNPase immunoreactivity (i.e., brightness intensity of the immunostain as measured by luminosity units) between the minocycline-treated (green) vs. the vehicle-treated (black) rats (n = 8 rats per group, with group average luminosity of 73.8 ± 3.5 luminosity units vs. 26.1 ± 1.9 luminosity units, respectively; P < 0.001, unpaired Student's t test). The histograms represent the distribution of total pixels (y axis) in the measured sections (480,000 pixels per section, 1 section per rat, and 8 sections per group for a total of 3.84 million pixels per group) of each group, plotted against luminosity units (x axis) where luminosity ranges from luminosity unit 0 (black) to 256 (white).

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