Mechanism-based inhibition of matrix metalloproteinase-9 provides neuroprotection in a mouse model of traumatic brain injury

Title from PDF of title page (University of Missouri--Columbia, viewed on September 19, 2012).The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Thesis advisor: Dr. Zezong GuIncludes bibliographical references.M.S. University of Missouri-Columbia 2012."May 2012"[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Traumatic brain injury (TBI) is a leading cause of death and disability in the U.S. Following an initial mechanical impact, TBI progresses to a secondary injury phase characterized by events that exacerbate brain damage. Matrix metalloproteinases (MMPs) are extracellular matrix-degrading enzymes that can cause detrimental outcomes via aberrant proteolysis, disruption of the blood-brain barrier (BBB), and neurodegeneration. In the present study we examined the roles of gelatinases (MMP-2/9) in an experimental mouse model of TBI, as well as the therapeutic potential of SB-3CT, a mechanism-based gelatinase inhibitor. Immunohistochemistry showed elevated nitrosative-oxidative stress within 24 hours of trauma. Gelatin zymography revealed that MMP-9 activity was significantly elevated during the first 7 days post-trauma. SB-3CT treatment for 7 days attenuated MMP-9 activity and thereby reduced histological lesion volumes, laminin degradation, and neuronal damage. Importantly, SB-3CT treatment improved long-term neurobehavioral outcomes in TBI mice, including motor function assessed by the beam-walking test, and spatial learning and memory using a Barnes maze. These results demonstrate considerable promise for the mechanism-based type of gelatinase inhibitors as a potential pharmacological treatment of brain injuries

Histological quantitation of brain injury using whole slide imaging: a pilot validation study in mice.

Quantitative assessment of serial brain sections provides an objective measure of neurological events at cellular and molecular levels but is difficult to implement in experimental neuroscience laboratories because of variation from person-to-person and the time required for analysis. Whole slide imaging (WSI) technology, recently introduced for pathological diagnoses, offers an electronic environment and a variety of computational tools for performing high-throughput histological analysis and managing the associated information. In our study, we applied various algorithms to quantify histologic changes associated with brain injury and compared the results to manual assessment. WSI showed a high degree of concordance with manual quantitation by Pearson correlation and strong agreement using Bland-Altman plots in: (i) cortical necrosis in cresyl-violet-stained brain sections of mice after focal cerebral ischemia; (ii) intracerebral hemorrhage in ischemic mouse brains for automated annotation of the small regions, rather than whole hemisphere of the tissue sections; (iii) Iba1-immunoreactive cell density in the adjacent and remote brain regions of mice subject to controlled cortical impact (CCI); and (iv) neuronal degeneration by silver staining after CCI. These results show that WSI, when appropriately applied and carefully validated, is a highly efficient and unbiased tool to locate and identify neuropathological features, delineate affected regions and histologically quantify these events

Comparison of the manual and automated annotations of intracerebral hemorrhage.

<p>The CV-stained coronal sections after focal cerebral ischemia in mice were used to examine intracerebral hemorrhage. The outlines in red and green indicate the manual annotations of the hemorrhagic areas and the regions chose for automated algorithm analysis, respectively. When the small regions were analyzed (<b>A</b>, <b>B</b>), the two annotations showed a high degree of concordance (<b>C</b>; R = 0.943, P = 0.000, n = 30), and the Bland-Altman difference plots (<b>D</b>) indicated that the automated annotations were consistently lower than the manual annotations. The arrow (<b>A, B,</b> embedded) shows that gaps between areas of blood cells, which were not excluded by manual measurement. When the whole hemisphere of the sections with hemorrhage were analyzed (<b>E, F; G</b> and <b>H</b> show an enlarged subregion of <b>E</b> and <b>F</b>, respectively), the Pearson correlation coefficient (<b>I</b>; R = 0.335, P = 0.003, n = 75) and Bland-Altman difference plots (<b>J</b>) showed low concordance and large difference between the manual and automated annotations. The red lines (in <b>D</b> and <b>J</b>) indicate mean and ±1.96 standard deviation. A, B, G, H: scale bar = 100 μm; E, F: scale bar = 2 mm.</p

Algorithm-assisted analysis of neurodegeneration on silver stained CCI sections.

<p>Brain sections at Bregma −2.02 mm from mice subjected to CCI were processed with silver staining to reveal neuronal degeneration (<b>A</b>). The green and red outlines indicate the two ROI we chose: ipsilateral cerebral peduncle (<b>B</b>) and a 200×200 μm² subregion (<b>D</b>) in the rostral region of the cerebral peduncle. Application of color deconvolution algorithm resulted in generation of mark-up images (<b>C</b>, <b>E</b>), and the optical density (OD) multiplied by the percentage of positive staining (%Pos) of these two regions were achieved, separately. Although the Pearson correlation coefficient (<b>F</b>) showed a high degree of concordance (R = 0.884, P = 0.000, n = 12) between OD × %Pos of both regions, the Bland-Altman difference plots (<b>G</b>) indicated that the data of the subregions were always higher than that of the whole cerebral peduncle. A: scale bar = 2 mm; B, C: scale bar = 200 μm; D, E: scale bar = 100 μm.</p

Comparison of the manual and automated annotations of microglial cell density in CCI sections.

<p>Iba-1 IHC staining was performed on the brain sections of mice subjected to CCI. A 640×480 μm² subregion at the peri-lesion cortex at the section of Bregma −1.14 mm and the cerebral peduncle at the section of Bregma −2.10 mm were used as two ROIs (green outline, <b>A</b> and <b>F</b>). The Iba-1 positive microglia within the cortex have a bushy morphology with thick, densely labeled processes and large cell bodies (<b>B</b>, embedded). The algorithm produced the mark-up images (<b>C</b>), where the red, orange, and yellow pixels visualize immunoreactivity-positive cells (strong, moderate, and weak intensity, respectively), whereas blue pixels depict immunoreactivity-negative cells. Pearson correlation coefficient (<b>D</b>) and Bland-Altman difference plots (<b>E</b>) showed a high degree of concordance (R = 0.756, P = 0.004, n = 12) and strong agreement between the manual and automated annotations of microglial cell density in cortex. However, the morphology of active microglia in the cerebral peduncle was different to that in cortex, and showed an amoeboid appearance with fewer processes (<b>G</b>, embedded). With adjustment of the algorithm parameters (<b>H</b>), Pearson correlation coefficient (<b>I</b>) and Bland-Altman difference plots (<b>J</b>) showed high a degree of concordance (R = 0.838, P = 0.001, n = 12) and strong agreement between the manual and automated annotations of microglial cell density in cerebral peduncle. A, F: scale bar = 2 mm; B, C, G, H: scale bar = 200 μm.</p

Comparison of manual annotation and Genie classification of cortical necrosis.

<p>After transient focal cerebral ischemia in mice, the cresyl-violet (CV)-stained brain sections were analyzed (<b>A, C</b>). For each tested region, the outlines in green, red and blue indicate manual annotations of the ischemic cortex, the non-ischemic contralateral cortex, and the cortical necrosis area, respectively. The Genie classification algorithm recognized necrotic (pink) and intact (yellow) areas within the ischemic cortex (<b>B, D</b>). When the contralateral intact cortices were analyzed, the Genie classification algorithm indicated 3.39%±0.61% (n = 18) FPR (<b>E</b>). As the manually annotated necrosis areas were analyzed by Genie algorithm, it revealed 95.99%±0.55% (n = 21) positive recognition rate (<b>F</b>). Pearson correlation coefficient between these two annotations (<b>G</b>; R = 0.957, P = 0.000, n = 32), and Bland-Altman difference plots (<b>H</b>) comparing the agreement of two measurements are shown. The red lines indicate mean and ±1.96 standard deviation. A, B, E, F: scale bar = 2 mm; C, D: scale bar = 200 μm.</p

Selective Inhibition of Matrix Metalloproteinase-9 Attenuates Secondary Damage Resulting from Severe Traumatic Brain Injury

<div><p>Traumatic brain injury (TBI) is a leading cause of death and long-term disability. Following the initial insult, severe TBI progresses to a secondary injury phase associated with biochemical and cellular changes. The secondary injury is thought to be responsible for the development of many of the neurological deficits observed after TBI and also provides a window of opportunity for therapeutic intervention. Matrix metalloproteinase-9 (MMP-9 or gelatinase B) expression is elevated in neurological diseases and its activation is an important factor in detrimental outcomes including excitotoxicity, mitochondrial dysfunction and apoptosis, and increases in inflammatory responses and astrogliosis. In this study, we used an experimental mouse model of TBI to examine the role of MMP-9 and the therapeutic potential of SB-3CT, a mechanism-based gelatinase selective inhibitor, in ameliorating the secondary injury. We observed that activation of MMP-9 occurred within one day following TBI, and remained elevated for 7 days after the initial insult. SB-3CT effectively attenuated MMP-9 activity, reduced brain lesion volumes and prevented neuronal loss and dendritic degeneration. Pharmacokinetic studies revealed that SB-3CT and its active metabolite, <i>p</i>-OH SB-3CT, were rapidly absorbed and distributed to the brain. Moreover, SB-3CT treatment mitigated microglial activation and astrogliosis after TBI. Importantly, SB-3CT treatment improved long-term neurobehavioral outcomes, including sensorimotor function, and hippocampus-associated spatial learning and memory. These results demonstrate that MMP-9 is a key target for therapy to attenuate secondary injury cascades and that this class of mechanism-based gelatinase inhibitor–with such desirable pharmacokinetic properties–holds considerable promise as a potential pharmacological treatment of TBI.</p></div

Protective effect of SB-3CT against long-term hippocampal damage and cognitive deficits after TBI.

<p><b><i>A.</i></b> Quantification of hippocampal lesion volume in vehicle and SB-3CT-treated mice, 30 days post-trauma. **, p<0.01 by one-tailed, unpaired Student’s t-test; n = 7 in vehicle-treated, 8 in SB-3CT-treated mice. Data are expressed as means ± SEM. <b><i>B.</i></b> Barnes maze acquisition. Testing consisted of 10 trials (2 trials/day) over 5 days. Latency (sec) of each trial was measured. Two-way repeated-measures ANOVA revealed a significant interaction (p = 0.0149) and significant main effects of days (p<0.0001) and groups (p = 0.0011). SB-3CT treated mice performed significantly better than vehicle-treated mice on days 22 and 23 after TBI; *, p<0.05, **, p<0.01; n = 11 in sham, 10 in vehicle-treated, and 11 in SB-3CT-treated mice. Data are expressed as means ± SEM. <b><i>C.</i></b> For analysis of memory acquisition in the maze, the latency AUC over 10 trials was calculated for each animal, and group comparisons were performed by one-way ANOVA, Dunnett’s post test, showing that SB-3CT ameliorates cognitive deficits after TBI; *, p<0.05 vs. vehicle-treated; n = 11 in sham, 10 in vehicle-treated, and 11 in SB-3CT-treated mice. Data are expressed as means ± SEM. <b><i>D.</i></b> Correlation between hippocampal damage and memory deficits. Hippocampal lesion volumes in 30 days and AUC were correlated by one-tailed Pearson correlation test. Pearson r = 0.5817, p<0.01; n = 6 in sham, 6 in vehicle-treated, and 8 in SB-3CT-treated mice. These data indicated significant correlation and three groups: sham, vehicle-treated, and SB-3CT treated showed good separation.</p

Concentrations and pharmacokinetic parameters of SB-3CT and <i>p</i>-OH SB-3CT after repeated i.p. administration of SB-3CT to mice.

a<p>Concentrations in µM in plasma and in pmole/mg tissue in brain; <i>AUC</i> in µM·minutes in plasma and in pmole·minutes/mg in brain.</p>b<p>NQ = non-quantifiable.</p>c<p>NC = not calculated; the low levels observed did not allow for the calculation of the terminal half-life and <i>AUC</i>_{0–∞<b>.</b>}</p