- Email: [email protected]
Quantitative analyses of matrix metalloproteinase activity after traumatic brain injury in adult rats
BR A I N R ES E A RC H 1 2 8 0 ( 2 00 9 ) 1 7 2 –17 7
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Quantitative analyses of matrix metalloproteinase activity after traumatic brain injury in adult rats Takuro Hayashi a , Yuji Kaneko a , SeongJin Yu a , EunKyung Bae a , Christine E. Stahl b , Takeshi Kawase c , Harry van Loveren a , Paul R. Sanberg a , Cesar V. Borlongan a,⁎ a
Department of Neurosurgery, University of South Florida, 12906 Bruce B. Downs Blvd MDC78, Tampa, FL 33612, USA Department of Internal Medicine, Dwight D. Eisenhower Army Medical Center, 300 Hospital Road, Fort Gordon, Augusta, GA 30905-5650, USA c Department of Neurosurgery, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
Recent laboratory evidence implicates matrix metalloproteinases (MMPs) as playing a
Accepted 12 May 2009
pivotal role in ischemic and traumatic brain injuries (TBI). Here, quantitative real-time
Available online 21 May 2009
PCR analyses revealed that brains from TBI rats displayed significantly elevated MMP-9 expression at 24 h post-TBI, which remained upregulated at least until 48 h after injury.
Keywords:
Immunohistochemical analyses similarly revealed significantly increased MMP-9
Head injury
immunoreactivity at 24 and 48 h post-TBI. These results demonstrate that alterations
MMP-9
in MMPs (i.e., MMP-9) commenced immediately after TBI, suggesting that treatment
Cortex
strategies designed to maintain MMP integrity should be initiated in the acute phase of
Immunohistochemistry
injury.
Quantitative real-time PCR
1.
Introduction
Traumatic brain injury (TBI) is a serious public health problem in the United States, with annual estimates of 5 million new head injuries and 2 million succumbing to life-long difficulties in daily activities. The economic cost for TBI is $56 billion annually (Mammis et al., 2008). The last 2 decades witnessed an increase in TBI cases due to combat injuries, including blast, impact or acceleration/deceleration injuries to the head, sustained in Iraq and Afghanistan may result in TBI characterized by damaged to the frontal and temporal lobes (Chuck, 2008; Inglese et al., 2005; Kraus et al., 2007; Suh et al., 2006). Unfortunately, there is currently no proven effective therapy for TBI. Accordingly, based on the significant
© 2009 Elsevier B.V. All rights reserved.
economic burden and the lack of therapies, urgent research is warranted to elucidate TBI pathophysiology and its treatment. Animal models of TBI have recently focused on two reproducible techniques namely fluid percussion and controlled cortical impact (CCI) injury models (LaPlaca et al., 2007; Tehranian et al., 2008). The brain injury produced by the CCI model replicates many clinical pathologic features of TBI, including an initial necrotic cell death in the underlying cortical tissue and white matter axonal injury, followed by an apoptotic cell death in surrounding tissue due to multiple subsequent events such as edema, ischemia, excitotoxicity and altered gene expression (Dikranian et al., 2008; Riess et al., 2002; Sandhir et al., 2008; You et al., 2008).
⁎ Corresponding author. Fax: +1 813 974 3078. E-mail address: [email protected] (C.V. Borlongan). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.05.040
BR A I N R ES E A RC H 1 2 8 0 ( 2 00 9 ) 1 7 2 –1 77
Recent experimental studies have suggested the participation of matrix metalloproteinases (MMPs) in TBI (Falo et al., 2006, 2008; Hu et al., 2008). Indeed, elevated MMP-9 levels have been detected in the plasma or serum of TBI patients (Suehiro et al., 2004; Vajtr et al., 2008; Vilalta et al., 2008) and in the cortex and hippocampus of TBI animals (Falo et al., 2006, 2008; Kim et al., 2005; Truettner et al., 2005). In addition, treatment strategies for abrogating the TBIinduced MMP destabilization have been examined, such
173
as hypothermia and hyperbaric oxygen therapy, with encouraging outcomes (Hu et al., 2008; Truettner et al., 2005; Vlodavsky et al., 2006). In order to enhance the success of therapeutic interventions directed at MMPs, elucidating the temporal acute profile of MMP activity may prove beneficial in guiding treatment initiation. Thus, the present study employed quantitative analyses of MMP-9 expression using quantitative real-time PCR verified with routine immunohistochemistry.
Fig. 1 – QRT-PCR analyses of MMP-9 expression in TBI brains. (Panel A) Confirms RNA integrity under UV light by visualization of 28S- and 18S-rRNA bands on a denaturing gel containing ethidium bromide. (Panel B) A logarithmic plot of fluorescence signal, in triplicates, for MMP-9 and GAPDH mRNA expression in the brain post-TBI. Threshold cycle (Ct) values were calculated by the equation DDCt = ΔCtMMP-9 − ΔCtGAPDH where ΔCt is the difference in Ct values between MMP-9 and the GAPDH. (Panel C) QRT-PCR analyses of MMP-9 gene expression in the injured hemispheres (n = 3–5 from triplicate independent experiments) were performed 1, 8, 24, and 48 h after brain injury. Bars represent mean values ± SE. Asterisks indicate statistical significance: *p < 0.05 versus control.
174
BR A I N R ES E A RC H 1 2 8 0 ( 2 00 9 ) 1 7 2 –17 7
2.
Results
2.1.
TBI upregulates mRNA expression of MMP-9
For quantitative real-time PCR, RNA integrity was initially confirmed under UV light by visualization of 28S- and 18S-rRNA bands on a denaturing gel containing ethidium bromide (Fig. 1A). The relative quantity of mRNA expression in right injured hemisphere (i.e., TBI target region) was compared with control hemisphere (i.e., sham operated, intact right hemisphere) (Fig. 1B). ANOVA revealed significant timing effects for MMP-9 mRNA expressions after TBI (p's < 0.01) (Fig. 1C). The levels of
MMP-9 mRNA expression as revealed by QRT-PCR were not altered at 1 h post-TBI, then slightly increased at 8 h post-TBI (but did not reach statistical significance compared to sham controls, p's > 0.05), and significantly increased at 24 h and 48 h post-TBI (p < 0.05 versus sham controls). QRT-PCR revealed four-fold and ten-fold increments in MMP-9 levels at 24 h and 48 h compared to controls.
2.2. TBI induces MMP-9 accumulation in cortical tissue adjacent to impacted cortex In complete agreement with differential mRNA expressions of MMP-9 after TBI, immunofluorescent microscopic exami-
Fig. 2 – Immunohistochemical analyses of MMP-9 expression in cortex post-TBI. (Panel A) Sporadic MMP-9 positive cells were found in the cortex at all time points post-sham procedure. On the other hand, MMP-9 positive cells populated the cortex at 24 and 48 h post-TBI (scale bars= 50 μm). (Panel B) Quantitative analyses revealed statistical differences in MMP-9 immunoreactivity over time. Bar graphs represent mean values ± SE. Asterisks (versus 1 h and 8 h, or between 24 h and 48 h) indicate statistical significance at p < 0.05 (n = 3–5). (Panel C) Cell counts were performed by calculating the ratio of MMP-9 positive cells (green) double-labeled with Hoechst 33258 (blue) over total number of Hoechst positive cells. Arrow corresponds to MMP-9 (green) double-labeled with Hoechst, while arrowheads point to MMP-9 negative, but Hoechst positive cells at 48 h post-TBI (scale bars= 10 μm).
BR A I N R ES E A RC H 1 2 8 0 ( 2 00 9 ) 1 7 2 –1 77
nation revealed a significant increase in MMP-9 immunoreactive cells in the cortical tissue immediately adjacent to the TBI impacted cortex at 24 h and 48 h after injury compared to sham controls (Fig. 2). ANOVA also revealed significant timing effects on MMP-9 immunoreactivity following TBI induction (p's < 0.01). Few MMP-9 immunoreactive cells were detected at 1 h and 8 h post-TBI in the cortical tissue next to the TBI impacted cortex (p's > 0.05). By 24 h post-TBI, this cortical area adjacent to the TBI impacted cortex displayed a significant surge in MMP-9 immunoreactive cells, which became more pronounced by 48 h post-TBI compared to sham controls (p's < 0.05).
3.
Discussion
The present study demonstrates a dramatic elevation in MMP activity in the acute phase of TBI as revealed by both mRNA and immunohistochemical analyses of MMP-9. These data suggest that alterations in MMP closely approximate the host tissue reorganization that occurs immediately after the impact injury. Such increased MMP-9 mRNA expression (by 24 h post-TBI), with enhanced immunoreactivity reflected in the surrounding cortex adjacent to the TBI impacted cortex indicates that MMP destabilization spreads to the neighboring brain areas within hours. This acute injury profiling of gene and protein expression offers insights into the optimal timing for initiating treatment strategies designed to retard or prevent TBI-induced dysfunctions in MMPs. The maintenance of MMP integrity may represent a pathway for neuroprotection. Accumulating laboratory evidence implicates a pivotal role for MMPs in TBI (Falo et al., 2006, 2008; Hu et al., 2008). TBI patients exhibited elevated MMP-9 levels in their plasma or serum (Suehiro et al., 2004; Vajtr et al., 2008; Vilalta et al., 2008). In parallel, TBI animals displayed increased MMP-9 immunoreactivity in the cortex and hippocampus ipsilateral to the impacted cerebral hemisphere (Falo et al., 2006, 2008; Kim et al., 2005; Truettner et al., 2005). Treatment strategies, such as hypothermia and hyperbaric oxygen therapy, have been proven effective in blocking TBI-induced MMP destabilization (Hu et al., 2008; Truettner et al., 2005; Vlodavsky et al., 2006). However, it appears that only MMP-9, but not MMP-2, responds positively to both these treatment interventions (Truettner et al., 2005; Vlodavsky et al., 2006). Of note, hypothermia was initiated at 30 min after TBI and maintained for the next 4 h (Truettner et al., 2005), whereas hyperbaric oxygen therapy was conducted twice a day (45-minute sessions with a 5-minute interval between sessions; Vlodavsky et al., 2006) starting at 3 h post-injury. Based on the present data, the treatment initiation and timing of regimen of hypothermia and hyperbaric oxygen therapy may not be optimal since they did not correspond to the onset of MMP-9 alterations. It is likely that the therapeutic window for targeting MMPs in TBI appears critical if stable functional recovery is desired. The logical timing post-TBI for MMP normalization is to coincide treatment intervention with the onset of MMP destabilization. Based on the present mRNA and immunohistochemical phenotypic expression of MMP-9, the 24-hour period after injury corresponds to the maximal upregulation of this MMP which may represent as an
175
appropriate timeframe to abrogate MMP degenerative effects. That suppressing MMP expression is key to endogenous neuroprotection concurs with recent studies demonstrating that blockade of MMP is necessary for brain remodeling after ischemic brain injury (Lee et al., 2009; Murata et al., 2008). Because cerebral ischemia and TBI share many common cell death pathways, neuroprotective strategies that work in stroke, such as blocking MMP degradation, may also prove beneficial in TBI. Indeed, MMPs can be targeted therapeutically as revealed by findings demonstrating that TBI outcome is altered by additional or deletion of MMPs (Mori et al., 2002; Sifringer et al., 2007; Suehiro et al., 2004; Wang et al., 2000). In summary, our study provides further evidence of MMP involvement in the progression of TBI. The quantitative data generated by the present QRT-PCR analyses support previous studies characterizing aberrant MMP accumulation in TBI patients (Suehiro et al., 2004; Vajtr et al., 2008; Vilalta et al., 2008) and animals (Falo et al., 2006, 2008; Kim et al., 2005; Truettner et al., 2005). The observed upregulation of MMP-9 within the acute post-injury period likely corresponds to the optimal therapeutic window for commencing neuroprotective modalities against MMP dysfunction which may prove effective in treating TBI.
4.
Experimental procedures
4.1.
Surgical procedures
All animal procedures followed approved IACUC guidelines for use of animals in research. Ten-week old Sprague–Dawley rats (n = 8) were subjected to TBI using a controlled cortical impactor (Pittsburgh Precision Instruments, Inc, Pittsburgh, PA). Animals initially received buprenorphine (0.05 mg/kg, s.c.) at the time of anesthesia induction (ketamine, 100 mg/kg, i.p., mixed with 5 mg/kg xylazine, i.p.). Once deep anesthesia was achieved (by checking for pain reflexes), individual animals were fixed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). After exposing the skull, craniectomy (4 mm respectively) was performed over the right frontoparietal cortex (0.5 mm anterior and +2.8 mm lateral to the midline). The pneumatically operated TBI device (diameter = 3 mm) impacted the brain at a velocity of 6.0 m/s reaching a depth of 2.0 mm below the dura matter layer and remained in the brain for 150 ms. The impactor rod was angled 15° to the vertical to maintain a perpendicular position in reference to the tangential plane of the brain curvature at the impact surface. A linear variable displacement transducer (Macrosensors, Pennsauken, NJ), which was connected to the impactor, measured velocity and duration to verify consistency. Bone wax was used to cover the craniectomized region and the skin incision sutured thereafter. Sham injury surgeries (i.e., uninjured controls; n = 8) consisted of animals exposed to anesthesia, scalp incision, craniectomy, and suturing. A computer operated thermal blanket pad and a rectal thermometer allowed maintenance of body temperature within normal limits. All animals were closely monitored until recovery from anesthesia and over the next 48 h. Animals were randomly euthanized between 1 h and 48 h after TBI.
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4.2. Quantitative real-time PCR analysis (QRT-PCR) of MMP-9 gene expression Rats were randomly euthanized between 1 h and 48 h post-TBI using carbon dioxide overdose. The rats were perfused transcardially with 400 ml of cold phosphate-buffered saline (PBS). Both injured right hemispheres and sham right hemispheres were instantaneously frozen in liquid nitrogen, and stored at − 80 °C until processing for molecular assays. Total RNA was extracted from frozen brain using mirVana™ miRNA isolation kit (Ambion, St. Austin, TX) according to the manufacturer's instructions and the A260/280 ratio of RNA extraction corresponded to 2.2 which is considered high quality. For cDNA synthesis, total RNA (2 μg) was reverse-transcribed in a 20 μl volume of reaction mixture, using a RETROscript (Ambion) according to the manufacturer's instructions. Transcript reactions without the reverse transcriptase enzyme were performed for negative controls in subsequent PCR-reactions. The primer sequence of MMP-9 (MN047660) forward primer was 5′-TGCTCCTGGCTCTAGGCTAC-3′; reverse primer was 3′GCTTCTCTCCCATCATCTGG-3′. For quantitative MMP-9 gene expression, PCR amplification was performed in a each reaction mixture containing 300 ng cDNA sample, 200 nM of each primer, 0.1 Unit Taq DNA polymerase (Ambion), 200 μM dNTPs and 1.5 mM MgCl2 (total volume 25 μl) using mirVana™qRT-PCR miRNA detection kit (Ambion). The reaction mixture was heated at 94 °C for 3 min followed by 40 cycles, each consisting of incubation for 30 s at 94 °C, 40 s at 72 °C, and 30 s at 55 °C. Amplification and Sybr Green I (Applied Biosystems, Foster City, CA) detection were performed on iCycler iQ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Primers specific for housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to internal control for the amounts of cDNA generated from each samples. The primer sequences of GAPDH (NW047696) were as follows: forward, 5′-ATGGGAAGCTGGTCATCAAC-3′ and reverse 5′-GTGGTTCACACCCATCACAA-3′. For each gene, PCRs were run in triplicate, and the amplified transcripts were quantified using the comparative Ct method. Briefly, threshold cycle (Ct) values were calculated by the equation ΔΔCt = ΔCtMMP-9 − ΔCtGAPDH where ΔCt is the difference in Ct values between MMP-9 and the GAPDH. Xn can be calculated by the formula Xn = 2mean ΔΔCt. The differences between Ct values were less than 2%. The amplified PCR productions were electrophoresed on a 2% agarose gel containing 0.2 μg/μl ethidium bromide, and were confirmed the sequences using a state-of-the-art ABI 3730 XL 96-capillary sequencer. The productions were visualized under UV light and save digitally with Alphalmager 2000 (Alpha Innoteck Corporation, San Leandro, CA), and represented single and theoretical base paired band (data not shown). For all experiments, controls without template were incubated. Each primer pair was, when possible, designed to span an exon–exon boundary.
4.3.
Immunohistochemistry
Rats were randomly euthanized between 1 h and 48 h post-TBI using carbon dioxide overdose, and underwent transcardial perfusion with 200 ml of cold PBS and 200 ml of 4% paraformaldehyde in PBS. Brains were removed and post-
fixed in the same fixative overnight at 4 °C, and then embedded in paraffin. The brains were coronally sectioned at the thickness of 8 μm. All sections were mounted on glass slides for immunohistochemical examinations, and were first deparaffinized through 3 washes in xylene and rehydrated through graduated alcohol solution, followed by washing 3 times for 5 min in PBS. Sections were then incubated overnight at 4 °C with a mouse anti-matrix metalloproteinase-9 antibody (Abcam, Cambridge, MA, dilution 1:500) and washed 3 times in PBS. Then sections were incubated with secondary goat antimouse IgG-Alexa 488 (Invitrogen, Carlsbad, CA, dilution 1:1000) for 90 min. Finally sections were washed 3 times for 5 min each in PBS, then processed for Hoechst 33258 (bisBenzimideH 33258 trihydrochloride, Sigma, St. Louis, MO) for 30 min, washed in PBS, and cover-slipped with Fluoromount (Sigma). Control studies included exclusion of primary antibody substituted with 10% normal goat serum in PBS. No immunoreactivity was observed in these controls. For morphological analyses, immunoreactive cells in the cortex within the ipsilateral to the TBI hemisphere were examined using Axio Imager.Z1 (Carl Zeiss, Thornwood, NY). Specifically, 6 coronal sections at every 300 μm that approximately captured the entire damaged cortex (AP − 2.0 to +2.0 mm from the bregma) were examined from each rat and the number of positive cells was counted in each 6 high power fields and the averages were used for the statistical analyses.
4.4.
Statistical analyses
Repeated measures of ANOVA followed by posthoc test using Fisher's protected least significant difference (PLSD) was used to reveal statistical significance in both QRT-PCR and histological data. A statistically significant difference was pre-set at p < 0.05.
REFERENCES
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Kraus, M.F., Susmaras, T., Caughlin, B.P., Walker, C.J., Sweeney, J.A., Little, D.M., 2007. White matter integrity and cognition in chronic traumatic brain injury: a diffusion tensor imaging study. Brain 130, 2508–2519. LaPlaca, M.C., Simon, C.M., Prado, G.R., Cullen, D.K., 2007. CNS injury biomechanics and experimental models. Prog. Brain Res. 161, 13–26. Lee, H., Park, J.W., Kim, S.P., Lo, E.H., Lee, S.R., 2009. Doxycycline inhibits matrix metalloproteinase-9 and laminin degradation after transient global cerebral ischemia. Neurobiol. Dis. 2009. Jan 6. [Electronic publication ahead of print]. Mammis, A., McIntosh, T.K., Maniker, A.H., 2008. Erythropoietin as a neuroprotective agent in traumatic brain injury — review. Surg. Neurol. 2008. Sep 10. [Electronic publication ahead of print]. Mori, T., Wang, X., Aoki, T., Lo, E.H., 2002. Downregulation of matrix metalloproteinase-9 and attenuation of edema via inhibition of ERK mitogen activated protein kinase in traumatic brain injury. J. Neurotrauma 19, 1411–1419. Murata, Y., Rosell, A., Scannevin, R.H., Rhodes, K.J., Wang, X., Lo, E.H., 2008. Extension of the thrombolytic time window with minocycline in experimental stroke. Stroke 39, 3372–3377. Riess, P., Zhang, C., Saatman, K.E., Laurer, H.L., Longhi, L.G., Raghupathi, R., Lenzlinger, P.M., Lifshitz, J., Boockvar, J., Neugebauer, E., Snyder, E.Y., McIntosh, T.K., 2002. Transplanted neural cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury. Neurosurgery 51, 1043–1054. Sandhir, R., Onyszchuk, G., Berman, N.E., 2008. Exacerbated glial response in the aged mouse hippocampus following controlled cortical impact injury. Exp. Neurol. 213, 372–380. Sifringer, M., Stefovska, V., Zentner, I., Hansen, B., Stepulak, A., Knaute, C., Marzahn, J., Ikonomidou, C., 2007. The role of matrix metalloproteinases in infant traumatic brain injury. Neurobiol. Dis. 25, 526–535. Suehiro, E., Fujisawa, H., Akimura, T., Ishihara, H., Kajiwara, K., Kato, S., Fujii, M., Yamashita, S., Maekawa, T., Suzuki, M., 2004. Increased matrix metalloproteinase-9 in blood in association with activation of interleukin-6 after traumatic brain injury:
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influence of hypothermic therapy. J. Neurotrauma 21, 1706–1711. Suh, M., Kolster, R., Sarkar, R., McCandliss, B., Ghajar, J., 2006. Cognitive and neurobiological research consortium. Deficits in predictive smooth pursuit after mild traumatic brain injury. Neurosci. Lett. 401, 108–113. Tehranian, R., Rose, M.E., Vagni, V., Pickrell, A.M., Griffith, R.P., Liu, H., Clark, R.S., Dixon, C.E., Kochanek, P.M., Graham, S.H., 2008. Disruption of Bax protein prevents neuronal cell death but produces cognitive impairment in mice following traumatic brain injury. J. Neurotrauma 25, 755–767. Truettner, J.S., Alonso, O.F., Dalton Dietrich, W., 2005. Influence of therapeutic hypothermia on matrix metalloproteinase activity after traumatic brain injury in rats. J. Cereb. Blood Flow Metab. 25, 1505–1516. Vajtr, D., Benada, O., Kukacka, J., Prusa, R., Houstava, L., Toupalik, P., Kizek, R., 2008. Correlation of ultrastructural changes of endothelial cells and astrocytes occurring during blood brain barrier damage after traumatic brain injury with biochemical markers of BBB leakage and inflammatory response. Physiol. Res. 2008. Apr 1. [Electronic publication ahead of print]. Vilalta, A., Sahuquillo, J., Rosell, A., Poca, M.A., Riveiro, M., Montaner, J., 2008. Moderate and severe traumatic brain injury induce early overexpression of systemic and brain gelatinases. Intensive Care Med. 34, 1384–1392. Vlodavsky, E., Palzur, E., Soustiel, J.F., 2006. Hyperbaric oxygen therapy reduces neuroinflammation and expression of matrix metalloproteinase-9 in the rat model of traumatic brain injury. Neuropathol. Appl. Neurobiol. 32, 40–50. Wang, X., Jung, J., Asahi, M., Chwang, W., Russo, L., Moskowitz, M.A., Dixon, C.E., Fini, M.E., Lo, E.H., 2000. Effects of matrix metalloproteinase-9 gene knock-out on morphological and motor outcomes after traumatic brain injury. J. Neurosci. 15, 7037–7042. You, Z., Savitz, S.I., Yang, J., Degterev, A., Yuan, J., Cuny, G.D., Moskowitz, M.A., Whalen, M.J., 2008. Necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice. J. Cereb. Blood Flow Metab. 28, 1564–1573.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Quantitative analyses of matrix metalloproteinase activity after traumatic brain injury in adult rats Takuro Hayashi a , Yuji Kaneko a , SeongJin Yu a , EunKyung Bae a , Christine E. Stahl b , Takeshi Kawase c , Harry van Loveren a , Paul R. Sanberg a , Cesar V. Borlongan a,⁎ a
Department of Neurosurgery, University of South Florida, 12906 Bruce B. Downs Blvd MDC78, Tampa, FL 33612, USA Department of Internal Medicine, Dwight D. Eisenhower Army Medical Center, 300 Hospital Road, Fort Gordon, Augusta, GA 30905-5650, USA c Department of Neurosurgery, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
Recent laboratory evidence implicates matrix metalloproteinases (MMPs) as playing a
Accepted 12 May 2009
pivotal role in ischemic and traumatic brain injuries (TBI). Here, quantitative real-time
Available online 21 May 2009
PCR analyses revealed that brains from TBI rats displayed significantly elevated MMP-9 expression at 24 h post-TBI, which remained upregulated at least until 48 h after injury.
Keywords:
Immunohistochemical analyses similarly revealed significantly increased MMP-9
Head injury
immunoreactivity at 24 and 48 h post-TBI. These results demonstrate that alterations
MMP-9
in MMPs (i.e., MMP-9) commenced immediately after TBI, suggesting that treatment
Cortex
strategies designed to maintain MMP integrity should be initiated in the acute phase of
Immunohistochemistry
injury.
Quantitative real-time PCR
1.
Introduction
Traumatic brain injury (TBI) is a serious public health problem in the United States, with annual estimates of 5 million new head injuries and 2 million succumbing to life-long difficulties in daily activities. The economic cost for TBI is $56 billion annually (Mammis et al., 2008). The last 2 decades witnessed an increase in TBI cases due to combat injuries, including blast, impact or acceleration/deceleration injuries to the head, sustained in Iraq and Afghanistan may result in TBI characterized by damaged to the frontal and temporal lobes (Chuck, 2008; Inglese et al., 2005; Kraus et al., 2007; Suh et al., 2006). Unfortunately, there is currently no proven effective therapy for TBI. Accordingly, based on the significant
© 2009 Elsevier B.V. All rights reserved.
economic burden and the lack of therapies, urgent research is warranted to elucidate TBI pathophysiology and its treatment. Animal models of TBI have recently focused on two reproducible techniques namely fluid percussion and controlled cortical impact (CCI) injury models (LaPlaca et al., 2007; Tehranian et al., 2008). The brain injury produced by the CCI model replicates many clinical pathologic features of TBI, including an initial necrotic cell death in the underlying cortical tissue and white matter axonal injury, followed by an apoptotic cell death in surrounding tissue due to multiple subsequent events such as edema, ischemia, excitotoxicity and altered gene expression (Dikranian et al., 2008; Riess et al., 2002; Sandhir et al., 2008; You et al., 2008).
⁎ Corresponding author. Fax: +1 813 974 3078. E-mail address: [email protected] (C.V. Borlongan). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.05.040
BR A I N R ES E A RC H 1 2 8 0 ( 2 00 9 ) 1 7 2 –1 77
Recent experimental studies have suggested the participation of matrix metalloproteinases (MMPs) in TBI (Falo et al., 2006, 2008; Hu et al., 2008). Indeed, elevated MMP-9 levels have been detected in the plasma or serum of TBI patients (Suehiro et al., 2004; Vajtr et al., 2008; Vilalta et al., 2008) and in the cortex and hippocampus of TBI animals (Falo et al., 2006, 2008; Kim et al., 2005; Truettner et al., 2005). In addition, treatment strategies for abrogating the TBIinduced MMP destabilization have been examined, such
173
as hypothermia and hyperbaric oxygen therapy, with encouraging outcomes (Hu et al., 2008; Truettner et al., 2005; Vlodavsky et al., 2006). In order to enhance the success of therapeutic interventions directed at MMPs, elucidating the temporal acute profile of MMP activity may prove beneficial in guiding treatment initiation. Thus, the present study employed quantitative analyses of MMP-9 expression using quantitative real-time PCR verified with routine immunohistochemistry.
Fig. 1 – QRT-PCR analyses of MMP-9 expression in TBI brains. (Panel A) Confirms RNA integrity under UV light by visualization of 28S- and 18S-rRNA bands on a denaturing gel containing ethidium bromide. (Panel B) A logarithmic plot of fluorescence signal, in triplicates, for MMP-9 and GAPDH mRNA expression in the brain post-TBI. Threshold cycle (Ct) values were calculated by the equation DDCt = ΔCtMMP-9 − ΔCtGAPDH where ΔCt is the difference in Ct values between MMP-9 and the GAPDH. (Panel C) QRT-PCR analyses of MMP-9 gene expression in the injured hemispheres (n = 3–5 from triplicate independent experiments) were performed 1, 8, 24, and 48 h after brain injury. Bars represent mean values ± SE. Asterisks indicate statistical significance: *p < 0.05 versus control.
174
BR A I N R ES E A RC H 1 2 8 0 ( 2 00 9 ) 1 7 2 –17 7
2.
Results
2.1.
TBI upregulates mRNA expression of MMP-9
For quantitative real-time PCR, RNA integrity was initially confirmed under UV light by visualization of 28S- and 18S-rRNA bands on a denaturing gel containing ethidium bromide (Fig. 1A). The relative quantity of mRNA expression in right injured hemisphere (i.e., TBI target region) was compared with control hemisphere (i.e., sham operated, intact right hemisphere) (Fig. 1B). ANOVA revealed significant timing effects for MMP-9 mRNA expressions after TBI (p's < 0.01) (Fig. 1C). The levels of
MMP-9 mRNA expression as revealed by QRT-PCR were not altered at 1 h post-TBI, then slightly increased at 8 h post-TBI (but did not reach statistical significance compared to sham controls, p's > 0.05), and significantly increased at 24 h and 48 h post-TBI (p < 0.05 versus sham controls). QRT-PCR revealed four-fold and ten-fold increments in MMP-9 levels at 24 h and 48 h compared to controls.
2.2. TBI induces MMP-9 accumulation in cortical tissue adjacent to impacted cortex In complete agreement with differential mRNA expressions of MMP-9 after TBI, immunofluorescent microscopic exami-
Fig. 2 – Immunohistochemical analyses of MMP-9 expression in cortex post-TBI. (Panel A) Sporadic MMP-9 positive cells were found in the cortex at all time points post-sham procedure. On the other hand, MMP-9 positive cells populated the cortex at 24 and 48 h post-TBI (scale bars= 50 μm). (Panel B) Quantitative analyses revealed statistical differences in MMP-9 immunoreactivity over time. Bar graphs represent mean values ± SE. Asterisks (versus 1 h and 8 h, or between 24 h and 48 h) indicate statistical significance at p < 0.05 (n = 3–5). (Panel C) Cell counts were performed by calculating the ratio of MMP-9 positive cells (green) double-labeled with Hoechst 33258 (blue) over total number of Hoechst positive cells. Arrow corresponds to MMP-9 (green) double-labeled with Hoechst, while arrowheads point to MMP-9 negative, but Hoechst positive cells at 48 h post-TBI (scale bars= 10 μm).
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nation revealed a significant increase in MMP-9 immunoreactive cells in the cortical tissue immediately adjacent to the TBI impacted cortex at 24 h and 48 h after injury compared to sham controls (Fig. 2). ANOVA also revealed significant timing effects on MMP-9 immunoreactivity following TBI induction (p's < 0.01). Few MMP-9 immunoreactive cells were detected at 1 h and 8 h post-TBI in the cortical tissue next to the TBI impacted cortex (p's > 0.05). By 24 h post-TBI, this cortical area adjacent to the TBI impacted cortex displayed a significant surge in MMP-9 immunoreactive cells, which became more pronounced by 48 h post-TBI compared to sham controls (p's < 0.05).
3.
Discussion
The present study demonstrates a dramatic elevation in MMP activity in the acute phase of TBI as revealed by both mRNA and immunohistochemical analyses of MMP-9. These data suggest that alterations in MMP closely approximate the host tissue reorganization that occurs immediately after the impact injury. Such increased MMP-9 mRNA expression (by 24 h post-TBI), with enhanced immunoreactivity reflected in the surrounding cortex adjacent to the TBI impacted cortex indicates that MMP destabilization spreads to the neighboring brain areas within hours. This acute injury profiling of gene and protein expression offers insights into the optimal timing for initiating treatment strategies designed to retard or prevent TBI-induced dysfunctions in MMPs. The maintenance of MMP integrity may represent a pathway for neuroprotection. Accumulating laboratory evidence implicates a pivotal role for MMPs in TBI (Falo et al., 2006, 2008; Hu et al., 2008). TBI patients exhibited elevated MMP-9 levels in their plasma or serum (Suehiro et al., 2004; Vajtr et al., 2008; Vilalta et al., 2008). In parallel, TBI animals displayed increased MMP-9 immunoreactivity in the cortex and hippocampus ipsilateral to the impacted cerebral hemisphere (Falo et al., 2006, 2008; Kim et al., 2005; Truettner et al., 2005). Treatment strategies, such as hypothermia and hyperbaric oxygen therapy, have been proven effective in blocking TBI-induced MMP destabilization (Hu et al., 2008; Truettner et al., 2005; Vlodavsky et al., 2006). However, it appears that only MMP-9, but not MMP-2, responds positively to both these treatment interventions (Truettner et al., 2005; Vlodavsky et al., 2006). Of note, hypothermia was initiated at 30 min after TBI and maintained for the next 4 h (Truettner et al., 2005), whereas hyperbaric oxygen therapy was conducted twice a day (45-minute sessions with a 5-minute interval between sessions; Vlodavsky et al., 2006) starting at 3 h post-injury. Based on the present data, the treatment initiation and timing of regimen of hypothermia and hyperbaric oxygen therapy may not be optimal since they did not correspond to the onset of MMP-9 alterations. It is likely that the therapeutic window for targeting MMPs in TBI appears critical if stable functional recovery is desired. The logical timing post-TBI for MMP normalization is to coincide treatment intervention with the onset of MMP destabilization. Based on the present mRNA and immunohistochemical phenotypic expression of MMP-9, the 24-hour period after injury corresponds to the maximal upregulation of this MMP which may represent as an
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appropriate timeframe to abrogate MMP degenerative effects. That suppressing MMP expression is key to endogenous neuroprotection concurs with recent studies demonstrating that blockade of MMP is necessary for brain remodeling after ischemic brain injury (Lee et al., 2009; Murata et al., 2008). Because cerebral ischemia and TBI share many common cell death pathways, neuroprotective strategies that work in stroke, such as blocking MMP degradation, may also prove beneficial in TBI. Indeed, MMPs can be targeted therapeutically as revealed by findings demonstrating that TBI outcome is altered by additional or deletion of MMPs (Mori et al., 2002; Sifringer et al., 2007; Suehiro et al., 2004; Wang et al., 2000). In summary, our study provides further evidence of MMP involvement in the progression of TBI. The quantitative data generated by the present QRT-PCR analyses support previous studies characterizing aberrant MMP accumulation in TBI patients (Suehiro et al., 2004; Vajtr et al., 2008; Vilalta et al., 2008) and animals (Falo et al., 2006, 2008; Kim et al., 2005; Truettner et al., 2005). The observed upregulation of MMP-9 within the acute post-injury period likely corresponds to the optimal therapeutic window for commencing neuroprotective modalities against MMP dysfunction which may prove effective in treating TBI.
4.
Experimental procedures
4.1.
Surgical procedures
All animal procedures followed approved IACUC guidelines for use of animals in research. Ten-week old Sprague–Dawley rats (n = 8) were subjected to TBI using a controlled cortical impactor (Pittsburgh Precision Instruments, Inc, Pittsburgh, PA). Animals initially received buprenorphine (0.05 mg/kg, s.c.) at the time of anesthesia induction (ketamine, 100 mg/kg, i.p., mixed with 5 mg/kg xylazine, i.p.). Once deep anesthesia was achieved (by checking for pain reflexes), individual animals were fixed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). After exposing the skull, craniectomy (4 mm respectively) was performed over the right frontoparietal cortex (0.5 mm anterior and +2.8 mm lateral to the midline). The pneumatically operated TBI device (diameter = 3 mm) impacted the brain at a velocity of 6.0 m/s reaching a depth of 2.0 mm below the dura matter layer and remained in the brain for 150 ms. The impactor rod was angled 15° to the vertical to maintain a perpendicular position in reference to the tangential plane of the brain curvature at the impact surface. A linear variable displacement transducer (Macrosensors, Pennsauken, NJ), which was connected to the impactor, measured velocity and duration to verify consistency. Bone wax was used to cover the craniectomized region and the skin incision sutured thereafter. Sham injury surgeries (i.e., uninjured controls; n = 8) consisted of animals exposed to anesthesia, scalp incision, craniectomy, and suturing. A computer operated thermal blanket pad and a rectal thermometer allowed maintenance of body temperature within normal limits. All animals were closely monitored until recovery from anesthesia and over the next 48 h. Animals were randomly euthanized between 1 h and 48 h after TBI.
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4.2. Quantitative real-time PCR analysis (QRT-PCR) of MMP-9 gene expression Rats were randomly euthanized between 1 h and 48 h post-TBI using carbon dioxide overdose. The rats were perfused transcardially with 400 ml of cold phosphate-buffered saline (PBS). Both injured right hemispheres and sham right hemispheres were instantaneously frozen in liquid nitrogen, and stored at − 80 °C until processing for molecular assays. Total RNA was extracted from frozen brain using mirVana™ miRNA isolation kit (Ambion, St. Austin, TX) according to the manufacturer's instructions and the A260/280 ratio of RNA extraction corresponded to 2.2 which is considered high quality. For cDNA synthesis, total RNA (2 μg) was reverse-transcribed in a 20 μl volume of reaction mixture, using a RETROscript (Ambion) according to the manufacturer's instructions. Transcript reactions without the reverse transcriptase enzyme were performed for negative controls in subsequent PCR-reactions. The primer sequence of MMP-9 (MN047660) forward primer was 5′-TGCTCCTGGCTCTAGGCTAC-3′; reverse primer was 3′GCTTCTCTCCCATCATCTGG-3′. For quantitative MMP-9 gene expression, PCR amplification was performed in a each reaction mixture containing 300 ng cDNA sample, 200 nM of each primer, 0.1 Unit Taq DNA polymerase (Ambion), 200 μM dNTPs and 1.5 mM MgCl2 (total volume 25 μl) using mirVana™qRT-PCR miRNA detection kit (Ambion). The reaction mixture was heated at 94 °C for 3 min followed by 40 cycles, each consisting of incubation for 30 s at 94 °C, 40 s at 72 °C, and 30 s at 55 °C. Amplification and Sybr Green I (Applied Biosystems, Foster City, CA) detection were performed on iCycler iQ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Primers specific for housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to internal control for the amounts of cDNA generated from each samples. The primer sequences of GAPDH (NW047696) were as follows: forward, 5′-ATGGGAAGCTGGTCATCAAC-3′ and reverse 5′-GTGGTTCACACCCATCACAA-3′. For each gene, PCRs were run in triplicate, and the amplified transcripts were quantified using the comparative Ct method. Briefly, threshold cycle (Ct) values were calculated by the equation ΔΔCt = ΔCtMMP-9 − ΔCtGAPDH where ΔCt is the difference in Ct values between MMP-9 and the GAPDH. Xn can be calculated by the formula Xn = 2mean ΔΔCt. The differences between Ct values were less than 2%. The amplified PCR productions were electrophoresed on a 2% agarose gel containing 0.2 μg/μl ethidium bromide, and were confirmed the sequences using a state-of-the-art ABI 3730 XL 96-capillary sequencer. The productions were visualized under UV light and save digitally with Alphalmager 2000 (Alpha Innoteck Corporation, San Leandro, CA), and represented single and theoretical base paired band (data not shown). For all experiments, controls without template were incubated. Each primer pair was, when possible, designed to span an exon–exon boundary.
4.3.
Immunohistochemistry
Rats were randomly euthanized between 1 h and 48 h post-TBI using carbon dioxide overdose, and underwent transcardial perfusion with 200 ml of cold PBS and 200 ml of 4% paraformaldehyde in PBS. Brains were removed and post-
fixed in the same fixative overnight at 4 °C, and then embedded in paraffin. The brains were coronally sectioned at the thickness of 8 μm. All sections were mounted on glass slides for immunohistochemical examinations, and were first deparaffinized through 3 washes in xylene and rehydrated through graduated alcohol solution, followed by washing 3 times for 5 min in PBS. Sections were then incubated overnight at 4 °C with a mouse anti-matrix metalloproteinase-9 antibody (Abcam, Cambridge, MA, dilution 1:500) and washed 3 times in PBS. Then sections were incubated with secondary goat antimouse IgG-Alexa 488 (Invitrogen, Carlsbad, CA, dilution 1:1000) for 90 min. Finally sections were washed 3 times for 5 min each in PBS, then processed for Hoechst 33258 (bisBenzimideH 33258 trihydrochloride, Sigma, St. Louis, MO) for 30 min, washed in PBS, and cover-slipped with Fluoromount (Sigma). Control studies included exclusion of primary antibody substituted with 10% normal goat serum in PBS. No immunoreactivity was observed in these controls. For morphological analyses, immunoreactive cells in the cortex within the ipsilateral to the TBI hemisphere were examined using Axio Imager.Z1 (Carl Zeiss, Thornwood, NY). Specifically, 6 coronal sections at every 300 μm that approximately captured the entire damaged cortex (AP − 2.0 to +2.0 mm from the bregma) were examined from each rat and the number of positive cells was counted in each 6 high power fields and the averages were used for the statistical analyses.
4.4.
Statistical analyses
Repeated measures of ANOVA followed by posthoc test using Fisher's protected least significant difference (PLSD) was used to reveal statistical significance in both QRT-PCR and histological data. A statistically significant difference was pre-set at p < 0.05.
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