Original Article

Matrix metalloproteinases and their tissue

inhibitors in serum and cerebrospinal fluid
of patients with moderate and severe
traumatic brain injury
Kebin Zheng, Chunhui Li1, Xiaosong Shan1, Haipeng Liu1, Wufang Fan, Zhenshan Wang, Ping Zheng2

Department of Neuroscience, College of Life Science, Hebei University, 1Departments of Neurosurgery, Affiliated Hospital of Hebei
University, Baoding 071000, Hebei Province, 2Shanghai Pudong New area People’s Hospital, 201299, Shanghai, People’s Republic of
China

Address for correspondence:
Prof. Zhenshan Wang,
College of Life Science, Hebei
University, People’s Republic of China.
E‑mail: jiwen0008@126.com
Received: 02‑10‑2013
Review completed: 23‑11‑2013
Accepted: 09‑12‑2013
Abstract
Objective: In this study, we investigated matrix metalloproteinases (MMPs) and tissue inhibitor
of metalloproteinase (TIMPs) in cerebrospinal fluid (CSF) and plasma of traumatic brain
injury (TBI) patients. Patients and Methods: A total of 30 patients with moderate and severe
TBI and 15 age‑matched controls were enrolled in this study. Plasma and CSF samples were
collected within 24 h (as the initial value), at 72 and 120 h post injury. CSF and plasma
MMP‑9, MMP‑2, TIMP‑1 and TIMP‑2 were estimated using ELISA. Different levels of these
indexes were compared in the two groups and further investigated the correlation between
each other. Results: There was a significant elevation in the levels of the initial MMP‑9 in the
CSF (P < 0.05), which lasted for 72 h post injury. TIMP‑1 kept increasing within 120 h post
injury and it was different compared with TIMP‑1 at 24 and 72 h post injury. Plasma levels of
MMP‑9, MMP‑2, TIMP‑1 and TIMP‑2 in TBI patients were also significantly different from
those in controls. Furthermore the CSF MMP‑9 in patients with severe TBI was higher than
that in patients with moderate TBI. In addition, there was a positive relationship between the
initial MMP‑9 and TIMP‑1 at 120 h post injury (r = 0.614, P < 0.01). Conclusion: MMPs
and TIMPs are increased in both CSF and plasma of TBI patients. TIMP‑1 has a positive
correlation with MMP‑9 and the initial MMP‑9 is associated with the neurological outcomes.
Key words: Cerebrospinal fluid, matrix metalloproteinase, tissue inhibitor of
metalloproteinase, traumatic brain injury

Introduction public health problem.[2] The scenario is same in China

Traumatic brain injury (TBI) is a major cause of death
and disability in Western countries[1] and also a major
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DOI:
10.4103/0028-3886.125258
also.[3] The pathophysiological mechanisms implicated
in the cellular and molecular change following
TBI remains unclear. Reliable biomarkers for early
prediction of prognosis and functional recovery are
very few.[4,5] Matrix metalloproteinases (MMPs) are a
group of homologous enzyme activity dependent on
Ca2+ and Zn2+ in the medium protease family and these
participate in maintaining homeostasis of extracellular
matrix (ECM) and are associated with the destruction of
the blood‑brain barrier, cerebral edema, inflammatory
reactions and tissue necrosis.[6‑8] There is almost no
expression of MMP‑9 in normal brain tissues similar to

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 Zheng, et al.: MMPs and TIMPs in TBI

animals.[9‑11] In animal experiments MMP‑9 significantly Data are expressed as mean  ±  standard error of the
increased within the first 3  h following brain injury mean. Two paired groups were compared with t‑test and
reaching a peak at 24 h and lasted for nearly a week.[12] multiple groups were compared with one‑way ANOVA.
Compared with wild type mice, mice with knock‑out Furthermore, the relationship between the parameters
of MMP‑9 developed less autonomic dysfunction, less was tested by Pearson analysis. The receiver operating
brain injury[13] and also less MMP‑2 expression following characteristic (ROC) was used to assess the acute density
TBI.[14] Elevated levels of cerebrospinal fluid (CSF) and of MMP‑9 and MMP‑2 for the assessment. Significance
blood MMP‑9 were also found in TBI patients. Elevated was set as a P < 0.05.
levels of MMP‑2 were only detected in plasma at 72 h
post TBI, but not in the CSF.[15] In this study, we focused Results
on the CSF and plasma levels of MMP‑2, MMP‑9, tissue
inhibitor of metalloproteinase (TIMP‑1) and TIMP‑2 CSF MMP‑9, MMP‑2, TIMP‑1 and TIMP‑2 levels were
in patients with moderate and severe TBI. We also significantly elevated in TBI patients (measured within
investigated the relationship between these indexes and 24 h of trauma), compared with the control group
neurological outcome. (P  <  0.05) [Table 1]. Blood MMP‑9, MMP‑2, TIMP‑1,
TIMP‑2 levels in TBI patients within 24 h were also
Patients and Methods significantly different from those in controls (P < 0.05)
[Table 2].
The Hebei Medical University Human Research Review
Committee approved the study. Informed consent The CSF MMP‑9 level in patients with severe TBI was
was taken from the family members for inclusion in significantly higher than that in patients with moderate
the study. Patients selected were patients with TBI TBI (82.68 ± 17.22 vs. 42.42 ± 14.94). However, there were
within a 24 h window from insult to ventriculostomy,
performed for diagnostic and therapeutic purposes. Table 1: CSF MMP‑9, MMP‑2, TIMP‑1 and TIMP‑2 levels in TBI
patients and the control group
Thirty patients (21 males and 9 females, mean age
of 48 ± 6 years) with a moderate or severe head Group n MMP‑9 MMP‑2 TIMP‑1 TIMP‑2
TBI group 30 71.34±13.93 28.27±6.99 142.7±16.47 26.07+8.61
TBI (glasgow coma scale [GCS] score of 12 or less at
Control group 10 3.51±1.53 5.937±3.51 21.63±9.88 4.17±2.35
admission) and ventricular catheter were recruited P P<0.05 P<0.05 P<0.05 P<0.05
in the study between January 2012 and October 2012.
MMP ‑ Matrix metalloproteinases, TIMP ‑ Tissue inhibitor of metalloproteinase,
Patients with TBI with associated infection, peripheral TBI ‑ Traumatic brain injury
bleeding disorder, tumor and other concomitant
traumatic injuries were excluded. Patients were further Table 2: Plasma MMP‑9, MMP‑2, TIMP‑1 and TIMP‑2 levels in TBI
grouped into the moderate (GCS >8) or severe (GCS <9) patients and the control group
arm. 15 age and gender‑matched (10 males and Group n MMP‑9 MMP‑2 TIMP‑1 TIMP‑2
5 females with mean age of 47 ± 7 years) subjects with TBI group 30 68.01±7.84 13.28±22.79 133.2±20.21 25.57±4.74
acute headache, in whom diagnostic lumbar puncture Control group 10 2.51±1.13 0.22±0.09 63.65±9.86 5.17±2.75
was performed to exclude intracranial hemorrhage P P<0.05 P<0.05 P<0.05 P<0.05
or inflammatory diseases were the controls. Control MMP ‑ Matrix metalloproteinases, TIMP ‑ Tissue inhibitor of metalloproteinase,
TBI ‑ Traumatic brain injury
group had paired CSF and plasma collected at the time
of lumbar puncture.

Human MMP‑2, MMP‑9, TIMP‑1, TIMP‑2 Quantikine®

ELISA kits  (Shanghai, China) were used to measure
serum MMPs and TIMPs level. All patients with TBI
had paired samples at all‑time points and there were
no missing data.

All patients underwent Karnofsky assessment at

6 months by an experienced neurosurgeon who was
blinded to the laboratory data. A  score of 90‑100 was
considered as good outcome; while, a score  <90 was
considered as poor outcome.
Figure 1: The matrix metalloproteinase-9 reached the peak at 24 h post
injury and followed by a downward trend within 120 h after the brain
Statistical analyses were performed using the GraphPad injury; in contrast, tissue inhibitor of metalloproteinase-1 kept increasing
Prism software package (GraphPad 5.0, San Diego, CA). within 120 h following the injury

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 Zheng, et al.: MMPs and TIMPs in TBI

no significant differences in MMP‑2, TIMP‑1 and TIMP‑2
between the two groups [Table 3].
There was no significant difference in the serum
levels of MMP‑9, MMP‑2, TIMP‑1 and TIMP‑2
between severe TBI and moderate TBI, MMP‑9
(75.86 ± 41.50 vs. 62.00 ± 34.86), MMP‑2 (13.53 ± 17.34 vs.
13.10 ± 8.43), TIMP‑1 (138.61 ± 44.726 vs. 129.10 ± 37.27)
and TIMP‑2 (27.37, ±6.51 vs. 24.19 ± 20.63), respectively.
The CSF MMP‑9 level reached the peak within 24  h
post injury, which lasted at least 72 h and there was no
statistical difference between the 24‑h point (71.34 ± 13.93)
and 72‑h point (49.63 ± 8.93), but both of them were
different from the 120‑h point (26.15 ± 4.89). The peak
of TIMP‑1 occurred at 120‑h point (162.24 ± 17.15) and it
was much higher than that at 72‑h point (142.91 ± 11.17)
and 24‑h point (142.78 ± 16.47) [Figure 1]. However, CSF
levels of MMP‑2 and TIMP‑2 did not demonstrate any
significant change within 120 h post injury in patients
with TBI. Similarly, the blood levels of each factor had
no significant change over time.
In TBI patients, there was positive correlation between
CSF MMP‑9 (24 h) and CSF TIMP‑1 (120 h) (r = 0.614,
P  <  0.01), Figure  2. However, there were no other
correlations between these parameters at other time
points. Positive correlation between MMP‑2 and TIMP‑2
was found at each point of measurement [Table 4].
curve: 0.819 vs. 0.353). On the ROC curve for MMP‑9,
the corresponding specificity at 75.11 ng/mL was 100%
and the sensitivity was 64.7% [Figure 3].
Discussion
Several MMPs are constitutively expressed, including
MMP‑2, which is normally found in brain and CSF.
During inflammation and other acute brain injuries
MMPs are expressed.[16] In addition, MMP‑9 contains
a nuclear factor‑kappaB site,[17] which gets activation
during the inflammatory process. In acute stroke patients,
MMP‑9 levels increase in CSF and blood and also in the
blood of patients with intracranial hemorrhage.[18]
In our study, there was an increased CSF level of
TIMP‑1 and TIMP‑2 in TBI patients and further there
was a positive correlation between the initial MMP‑9
and TIMP‑1 at 120 h post injury. Other studies show the
TIMP‑1 can specifically inhibit the MMP‑9 and TIMP‑2
can selectively inhibit the MMP‑2.[19,20] The TIMP has a
Table 3: CSF MMP‑9, MMP‑2, TIMP‑1 and TIMP‑2 levels in
moderate and severe TBI patients
Group n MMP‑9 MMP‑2 TIMP‑1 TIMP‑2
Severe TBI 13 82.68±17.22 25.36±6.89 135.13±17.86 22.82±2.38
Moderate TBI 17 42.42±14.94 30.40±7.24 148.62±14.72 28.56±2.47
P P<0.05 P>0.05 P>0.05 P>0.05
MMP ‑ Matrix metalloproteinases, TIMP ‑ Tissue inhibitor of metalloproteinase,
TBI ‑ Traumatic brain injury

The average score in severe TBI group was 56.38 ± 15.64,

while that in moderate group was 78.91 ± 4.89. ROC Table 4: A positive correlation between MMP‑2 and TIMP‑2
curve, initial CSF MMP‑9 and MMP‑2 were correlate with Time point Correlation P
the long‑term quality of life assessment. Status of both 24 hours 0.475 0.008
72 hours 0.420 0.021
MMPs predicted the neurological outcome  [Figure  3].
120 hours 0.655 0.001
The MMP9 had a significant predictive value for
MMP ‑ Matrix metalloproteinases, TIMP ‑ Tissue inhibitor of metalloproteinase,
neurological outcome in TBI patients (area under the TBI ‑ Traumatic brain injury

Figure 2: The plot figure of initial matrix metalloproteinase (MMP9) and Figure 3: Comparison of receiver operating characteristic curves. Green
tissue inhibitor of metalloproteinase (TIMP1) at 120 h post injury. There curve: Probability of neurological outcome on matrix metalloproteinase
was a positive correlation between the initial MMP9 and TIMP1 on the 5th (MMP2) status; Blue curve: Probability of neurological outcome on
day post brain injury (r = 0.612, P < 0.05) MMP9 status

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 Zheng, et al.: MMPs and TIMPs in TBI
function of negative control in the metabolism of ECM,
which can inhibit the activation and function of MMPs
and further affect the protein proteolysis during the
injury.[21,22] Under pathological condition, the equilibrium
of MMPs and TIMPs is perturbed, then it will cause
a series of pathological changes implicated in several
neurological diseases.[23,24]
In this study, there was an increasing trend in CSF TIMP‑1
over a period of 120  h post injury. Both CSF MMP‑2
and TIMP‑2 did not show a consistent change during
the 120 h post injury, however, certain correlation was
maintained between them. We speculate that the changes
in TIMPs resulted from the activation of MMPs and
further the endogenous TIMPs might be neuroprotective.
This is further supported by the decreasing trend of
MMP‑9 with the progressive increase in TIMP‑1 and a
positive correlation between the initial MMP‑9 and later
TIMP‑1 post injury. This provides a theoretical basis for
exogenous TIMPs in TBI. In this study, admission CSF
MMP‑9 was a predictor of neurological outcome. This
further strengthens the interest of targeting MMPs in TBI.
There are some limitations to our study. The sample size
was small and the statistical power may not be robust.
The study focused on the MMP‑9 and MMP‑2, other
MMPs might be involved in the neuronal injury. In this
study, Karnofsky performance scale was used to assess
the outcome. However, this scale is much more focused
on the quality of life. It does not specifically assess the
sensori‑motor and cognitive dysfunction that occurs in
patients with TBI. In our future study, we would like to
enroll more patients to validate the observations made
in this study and further look at other MMPs and use
a battery of neurobehavioral and neurocognitive tests
to explore the exact correlation between MMPs and
neurological outcome in patients with TBI.
References
1. Bruns J Jr, Hauser WA. The epidemiology of traumatic brain injury:
A review. Epilepsia 2003;44:2‑10.
2. Langlois JA, Rutland‑Brown W, Wald MM. The epidemiology and impact
of traumatic brain injury: A brief overview. J Head Trauma Rehabil
2006;21:375‑8.
3. Jiang JY. Collaborators CHTS. Head trauma in China. Injury 2012;
In press.
4. Begaz T, Kyriacou DN, Segal J, Bazarian JJ. Serum biochemical
markers for post‑concussion syndrome in patients with mild traumatic
brain injury. J Neurotrauma 2006;23:1201‑10.
5. Kochanek PM, Berger RP, Bayir H, Wagner AK, Jenkins LW, Clark RS.
Biomarkers of primary and evolving damage in traumatic and ischemic
brain injury: Diagnosis, prognosis, probing mechanisms, and therapeutic
decision making. Curr Opin Crit Care 2008;14:135‑41.
6. Azeh I, Mäder M, Smirnov A, Beuche W, Nau R, Weber F. Experimental
pneumococcal meningitis in rabbits: The increase of matrix
metalloproteinase‑9 in cerebrospinal fluid correlates with leucocyte
invasion. Neurosci Lett 1998;256:127‑30.
7. Khrestchatisky M, Jourquin J, Ogier C, Charton G, Bernard A,
Tremblay E, et al. Matrix metalloproteinases and their inhibitors,
Neurology India | Nov-Dec 2013 | Vol 61 | Issue 6
modulators of neuro‑immune interactions and of pathophysiological
processes in the nervous system. J Soc Biol 2003;197:133‑44.
8. Kanoh Y, Ohara T, Kanoh M, Akahoshi T. Serum matrix
metalloproteinase‑2 levels indicate blood‑CSF barrier damage in
patients with infectious meningitis. Inflammation 2008;31:99‑104.
9. Rosenberg GA, Navratil M, Barone F, Feuerstein G. Proteolytic cascade
enzymes increase in focal cerebral ischemia in rat. J Cereb Blood Flow
Metab 1996;16:360‑6.
10. Gasche Y, Fujimura M, Morita‑Fujimura Y, Copin JC, Kawase M,
Massengale J, et al. Early appearance of activated matrix metalloproteinase‑9
after focal cerebral ischemia in mice: A possible role in blood‑brain barrier
dysfunction. J Cereb Blood Flow Metab 1999;19:1020‑8.
11. Heo JH, Lucero J, Abumiya T, Koziol JA, Copeland BR, del Zoppo GJ.
Matrix metalloproteinases increase very early during experimental focal
cerebral ischemia. J Cereb Blood Flow Metab 1999;19:624‑33.
12. Lin Y, Pan Y, Wang M, Huang X, Yin Y, Wang Y, et al. Blood‑brain
barrier permeability is positively correlated with cerebral microvascular
perfusion in the early fluid percussion‑injured brain of the rat. Lab
Invest 2012;92:1623‑34.
13. Wang X, Jung J, Asahi M, Chwang W, Russo L, Moskowitz MA, et al. Effects
of matrix metalloproteinase‑9 gene knock‑out on morphological and motor
outcomes after traumatic brain injury. J Neurosci 2000;20:7037‑42.
14. Wang X, Mori T, Jung JC, Fini ME, Lo EH. Secretion of matrix
metalloproteinase‑2 and‑9 after mechanical trauma injury in rat cortical
cultures and involvement of MAP kinase. J Neurotrauma 2002;19:615‑25.
15. Grossetete M, Phelps J, Arko L, Yonas H, Rosenberg GA. Elevation of
matrix metalloproteinases 3 and 9 in cerebrospinal fluid and blood in
patients with severe traumatic brain injury. Neurosurgery 2009;65:702‑8.
16. Johanson M, Zhao XR, Huynh‑Ba G, Villar CC. Matrix
metalloproteinases, tissue inhibitors of matrix metalloproteinases, and
inflammation in cyclosporine A‑induced gingival enlargement: A pilot
in vitro study using a three‑dimensional model of the human oral
mucosa. J Periodontol 2013;84:634‑40.
17. Wang JJ, Huan SK, Hsieh KH, Chou HC, Hsiao G, Jayakumar T,
et al. Inhibitory effect of midazolam on MMP‑9, MMP‑1 and MMP‑13
expression in PMA‑stimulated human chondrocytes via recovery of
NF‑κB signaling. Arch Med Sci 2013;9:332‑9.
18. Kavalci C, Genchallac H, Durukan P, Cevik Y. Value of biomarker‑based
diagnostic test in differential diagnosis of hemorrhagic‑ischemic stroke.
Bratisl Lek Listy 2011;112:398‑401.
19. Niebroj‑Dobosz I, Janik P, Sokołowska B, Kwiecinski H. Matrix
metalloproteinases and their tissue inhibitors in serum and cerebrospinal
fluid of patients with amyotrophic lateral sclerosis. Eur J Neurol
2010;17:226‑31.
20. Ahmed SH, Clark LL, Pennington WR, Webb CS, Bonnema DD,
Leonardi AH, et al. Matrix metalloproteinases/tissue inhibitors of
metalloproteinases: Relationship between changes in proteolytic determinants
of matrix composition and structural, functional, and clinical manifestations
of hypertensive heart disease. Circulation 2006;113:2089‑96.
21. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors
of metalloproteinases: Structure, function, and biochemistry. Circ Res
2003;92:827‑39.
22. Lorenzl S, Albers DS, LeWitt PA, Chirichigno JW, Hilgenberg SL,
Cudkowicz ME, et al. Tissue inhibitors of matrix metalloproteinases are
elevated in cerebrospinal fluid of neurodegenerative diseases. J Neurol
Sci 2003;207:71‑6.
23. Renaud S, Leppert D. Matrix metalloproteinases in neuromuscular
disease. Muscle Nerve 2007;36:1‑13.
24. Rosenberg GA. Matrix metalloproteinases and their multiple roles in
neurodegenerative diseases. Lancet Neurol 2009;8:205‑16.
How to cite this article: Zheng K, Li C, Shan X, Liu H, Fan W,
Wang Z, et al. Matrix metalloproteinases and their tissue inhibitors in
serum and cerebrospinal fluid of patients with moderate and severe
traumatic brain injury. Neurol India 2013;61:606-9.
Source of Support: Supported by the Natural Science Foundation
of Hebei Province (No. 2012201136) and Medical Science Special
Foundation of Hebei University  (No.  2012B2004), Conflict of
Interest: Hebei postdoctoral fund (No. 111867).
609
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