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Rice et al. Minocycline in spinal cord injury
[5]
and multiple sclerosis [1-3] . Minocycline has also that project through the spinal cord . These studies
been demonstrated to have beneficial effects in are relevant to the potential application of minocycline
the treatment of acute spinal cord injury (SCI), a as a neuroprotective agent after SCI.
condition for which therapeutic treatments are urgently
needed. Indeed, a phase 2 clinical trial administering METHODS
minocycline in acute human spinal cord injury was
completed in Canada with promising early results [4] Surgery and minocycline treatment
and a phase 3 clinical trial is ongoing (ClinicalTrials. CD1 outbred mice were anesthetized, the spinal cord
gov Identifier: NCT01828203). Encouragingly, the exposed, and extradural mechanical compression of
efficacy of minocycline in reducing tissue damage and the cord with a modified aneurysm clip was achieved
in ameliorating functional impairment in SCI has been as previously described . In brief, surgery consisted of
[5]
demonstrated despite variations in the models used, anesthetizing mice with a mixture of ketamine/xylazine
the species tested and the treatment paradigm [5-7] . (200 and 10 mg/kg, respectively) intraperitoneally.
The mechanism of action of minocycline in Animals were subsequently immobilized in a
neurological diseases appears not to be related to its stereotactic frame. An incision was made in the skin
antimicrobial activity, but rather to immunomodulation, and the muscle and tissue overlying the vertebral
blockade of excitotoxicity and inhibition of cell death column were blunt dissected away. Using the spiny
pathways [1,2,5-7] , all of which have been implicated in process of T2 as a landmark, a laminectomy was
the pathophysiology of SCI. performed at the level of T3/T4 and the spinal cord
was exposed. A rigid hook was used to clear a path
Minocycline has also been reported to inhibit the underneath the cord so that a modified aneurysm clip
production and activity [8-10] of a family of proteolytic with a closing force of 8 g could be applied. Extradural
enzymes known as the matrix metalloproteinases compression of the cord was achieved by allowing the
(MMPs). A large number of MMPs have been identified clip to slam shut on the cord producing mechanical
and, collectively, the MMPs are capable of degrading trauma. The clip was maintained in position for one
all components of the extracellular matrix (ECM). As minute producing damage that also had ischemic
such, MMPs have roles in several developmental components. Following injury the clip was removed
processes that involve ECM turnover as well as in and the wound was closed using nylon suture. Mice
wound healing throughout life. However, the aberrant recovered in a room maintained at 27 ºC where they
expression of these molecules is detrimental to several were kept thereafter for the determined survival time.
diseases of the CNS, including SCI [11] . We have Manual expression of the bladder was required twice
previously shown that several MMPs were upregulated daily and the food and water were placed directly in
after SCI in mice; in particular, we provided evidence the cage to allow ready access.
that MMP-12 expression after SCI was linked to
increased blood spinal barrier permeability, microglial One hour after surgery, mice were randomized and
activation, macrophage infiltration, and worsened injected intraperitoneally with either a solution of
functional outcome following injury [12] . In addition, minocycline at a dose of 50 mg/kg or saline vehicle
Hsu et al. [13] and Noble et al. [14] demonstrated that control. Subsequent injections were given at 24-h
MMP-9 limited functional recovery in the short-term intervals until sacrifice. Following the second injection
following SCI in rats, although MMP-2 had an opposite of 50 mg/kg, the dose of minocycline was reduced
outcome 7-14 days after injury. to 25 mg/kg for all remaining treatments. This dose
regimen is identical to that used in our previous study [5]
Given that minocycline is currently in a phase 3 that demonstrated behavioral recovery by 3 days post-
trial in traumatic SCI (ClinicalTrials.gov Identifier: injury in the minocycline compared to vehicle group.
NCT01828203), it is important to have a comprehensive
picture of its spectrum of activity. Thus, in this Analyses of MMPs
study, we determined whether or not the efficacy of TaqMan real time PCR was used to profile MMP
minocycline in SCI was due in part to its ability to mRNA levels as detailed [15] . Minocycline and vehicle
inhibit the expression or activity of MMPs. In addition, treated mice were sacrificed 1, 2 and 5 days after
we examined the response of microglia/macrophages injury (4/group) and 4 mice served as uninjured
following SCI and the impact of minocycline treatment. controls. Spinal cords were removed and a 1-cm
Finally, we addressed whether minocycline could segment including the injury site was homogenized
protect against the death of neurons at the site of in Trizol reagent and total RNA extracted. One
traumatic impact itself, since previous work has microgram of RNA was reverse transcribed to make
examined axons of passage and higher order neurons cDNA. Each PCR reaction contained the equivalent
244 Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ November 28, 2017