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Rice et al. Minocycline in spinal cord injury

of 5 ng of reverse transcribed RNA. The 18S rRNA were obtained for immunohistochemical analysis
gene was used as an endogenous internal control to of microglia, neurons and apoptotic cells using the
®
account for differences in the extraction of original antibodies Iba1, NeuN (Chemicon) and the Apoptag
tissue and reverse transcription of total RNA. Primer Fluorescein In Situ Apoptosis Detection Kit (Tunel,
sequences for the TaqMan assays were previously Chemicon). lba1 and NeuN immunolabeling to detect
described elsewhere [15] , and statistical validity for the microglia and neurons, respectively, was performed as
multiple TaqMan targets has been corroborated. previously described [12] . For NeuN labeling, sections
had to undergo antigen retrieval by boiling in 10 mmol/L
Gelatin zymography of one cm segments of spinal sodium citrate buffer (pH 6.5) for 10 min. Slides were
cord was described previously [16] as was the method incubated with mouse anti-NeuN (1:50, Chemicon)
of in situ zymography [17] . Note that while gelatin overnight at 4 ºC. Biotinylated anti-rabbit IgG was used
zymography is a technique reliant on the activity of for the secondary antibody and staining was visualized
MMP-2 and -9 to degrade gelatin in-gel, it is ultimately with ABC using DAB as the substrate.
proportional to, and thus a reflection of, the amount of
MMP-2 and -9 in the test samples. For blinded Iba1 analyses, the degree of microglial/
macrophage activation was determined by examining
While the above in-gel gelatin zymography is a the morphology and density of the Iba1 labeled
manifestation of protein content, the net gelatinolytic cells. Considerations were made for the size, shape
activity (i.e. overall enzyme activity in a milieu that and relative density of Iba1 labeled cells. Briefly,
also contains enzyme inhibitors) in an intact non- Iba1-stained sections were scored for microglial/
fixed tissue specimen can be evaluated using in situ macrophage activation using a scale from 0-4 where
zymography. For this method spinal cord injury was 0 was normal cord and 4 was the presence of highly
induced in 14 mice; 7 animals were injected with 50 activated microglia/macrophages. For NeuN labeled
mg/kg of minocycline 1 h and 24 h after, while the other tissue, longitudinal sections containing central canal
7 received saline vehicle at the same time points. One were identified as well as sections about 180 µm away
hour after the second injection, mice were given an on either side of the center section (total of 3 sections/
overdose of ketamine/xylazine intraperitoneally and the mouse). The number of positively stained cells was
spinal cord was carefully removed and directly frozen manually counted from 5 regions of each section
in isopentane at -70 ºC. Twenty µm thick longitudinal corresponding to lesion area, +1 mm rostral, +2 mm
sections were cut on a cryostat, mounted on glass rostral, +1 mm caudal and +2 mm caudal. Unbiased
slides and stored at -80 ºC. Sections were thawed and stereology was not used in the blinded counts. Data
incubated in a humid chamber in 100 mL (50 µL/s) of were analyzed using univariate analysis of variance
reaction buffer containing 100 mg/mL of FITC-labeled with scheffe post-hoc comparisons. Similarly, for Tunel
dye-quenched-gelatin (EnzCheck collagenase kit, labeled tissue, the total number of labeled cells in a
Molecular probes, Eugene, OR) for 3 h at 37 ºC [16,17] . 1-cm segment of cord, from a section containing
At the end of the incubation period slides were rinsed central canal and including the lesion epicenter,
in PBS, fixed and then mounted. Prepared slides were was counted. In this case, data was analyzed using
observed using fluorescence microscopy. Sections unpaired t-tests at 2 and 5 days.
incubated without dye-quenched-gelatin did not
exhibit fluorescence and served as negative controls. RESULTS
Samples from SCI animals were analyzed in a blinded
manner using a qualitative rating scale from 0 to 4, with Expression profile of MMP transcripts in
zero representing no fluorescence and a score of 4 minocycline and vehicle treated SCI animals
depicting maximal in situ zymography signal. The expression profile of 20 MMPs was examined by
TaqMan PCR in mouse spinal cord following injury.
Immunohistochemistry Figure 1 shows that transcripts encoding MMP-3, -7,
Spinal cord compression was performed in 36 mice -10, -11, -12, -13, -19, -20 and -21 were elevated after
as described above. Mice were randomly treated with spinal cord injury compared to uninjured controls,
either minocycline or vehicle and survived for 2 or 5 while those of MMP-23 and -24 were reduced.
days (n = 9/group). Animals were given an overdose of Compared to vehicle treated SCI samples, minocycline
ketamine/xylazine and the spinal cords were excised administration had no significant effect on altering the
and post-fixed in 10% neutral buffered formalin MMP transcripts at 1, 2 and 5 days after injury.
and subsequently embedded in paraffin wax. Six
micrometer thick sections were cut on a microtome We also measured transcripts encoding physiological
and collected at a frequency of 1:10. Three series antagonists of MMPs, the 4 tissue inhibitors of
Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ November 28, 2017 245