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Rice et al. Minocycline in spinal cord injury

Relative RNA level

P < 0.05
Relative RNA level

Figure 2: TIMPs remain largely unaltered by minocycline treatment after SCI. TIMP-1 was the only inhibitor that was altered (upregulated)
by injury compared to naïve controls, and there was no effect of minocycline treatment with the exception of an increase of TIMP-4 at 1
day after SCI. Values are mean ± SEM of 4 samples from vehicle (V) or minocycline (M) group, at 1, 2 or 5 days after injury. *P < 0.05,
**P < 0.01 and ***P < 0.001 compared to naïve controls (one-way ANOVA with Tukey’s multiple comparisons). TIMPs: tissue inhibitors of
metalloproteinases; SCI: spinal cord injury

metalloproteinases (TIMPs). Figure 2 shows that the MMP-2 species did not elevate profoundly after injury
expression of TIMP-1, but not TIMP-2, -3 and -4, was or in the presence of minocycline. The quantification of
upregulated by injury. The levels of all four TIMPs the gelatin zymograms is displayed in Figure 3B and
in the minocycline treated cords were not altered confirms the lack of effect of minocycline on MMP-2
from those of the vehicle treatment groups, with the and -9 protein expression after SCI.
exception of TIMP-4 that was raised by minocycline at
1 day of injury. In situ zymography reveals no alterations of
net proteolytic activity by minocycline
Gelatin zymography shows that minocycline Although gelatin zymography [Figure 3A] is based on
does not alter MMP-2 and -9 protein content the degradation of gelatin in-gel by MMP-2 and -9,
Measurement of transcripts encoding MMPs provides it is a reflection of the content of these gelatinases
a broad overview of the changes occurring to all (MMP-2 or -9) rather than their net enzymatic activity,
given that all enzyme co-factors are supplied in
known MMP members, since several individual MMPs optimal amounts for manifestation of catalysis in-gel.
continue to be difficult to measure using Western blot In addition, while the mRNA for MMPs are elevated
or activity assays. A reproducible and commonly used in SCI [Figure 1], so are the transcripts for the TIMP
method for protein levels involves the determination inhibitors [Figure 2], thus making it relevant to address
of MMP-2 and MMP-9 by the method of gelatin the balance of proteolytic activity in specimens.
zymography. Although we did not detect the elevation To determine the net proteolytic activity existent
of their transcripts after SCI [Figure 1], MMP-9 in the injured cord, in situ zymography was used,
protein can be made outside of the CNS, including in whereby the gelatin-FITC substrate was overlaid onto
neutrophils, and then deposited into the lesion site. For longitudinal unfixed sections encompassing the injured
these reasons, we examined the levels of MMP-2 and area [17] , and where if there is relative abundance
-9 protein using gelatin zymography. The pro-MMP-9 of enzyme over inhibitors, then there would be net
protein was minimally expressed in control uninjured proteolysis of the gelatin-FITC. In normal uninjured
spinal cord tissue [Figure 3A]. Injury resulted in an cord, no fluorescence signal was evident. Twenty-
upregulation of both the pro- and active forms of MMP- four hours following SCI, varying grades of signals
9 one day after injury. Minocycline treatment had no were observed [Figure 3C]. Blinded analyses across
obvious effect on the injury-induced MMP-9. The pro- 14 injured specimens concluded that there was no
Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ November 28, 2017 247