Local neutrophil influx following lateral fluid-percussion brain injury in rats is associated with accumulation of complement activation fragments of the third component (C3) of the complement system
Introduction
Traumatic brain injury (TBI) is one of the major causes of mortality in the United States among juveniles and young adults, with a median age of 25 years. The patients who survive are often faced with long-term neurological and socio-economic difficulties (Marshall et al., 1991, Stahel et al., 1998). The prolonged time course of the neuropathology seen in survivors has been attributed to the profound inflammatory response within the cranial cavity resulting in secondary neuronal death (Stahel et al., 1997, Holmin et al., 1998). Experimental studies have shown that early after injury, polymorphonuclear leukocytes are the first inflammatory cells to adhere to the vascular endothelium and migrate into the brain parenchyma through the disrupted blood brain barrier (BBB) (Schoettle et al., 1990, Soares et al., 1995, Holmin et al., 1998). These neutrophils are attracted across the BBB by the up-regulation of leukocyte adhesion molecules, chemoattractant mediators, and activated complement components (Kaczorowski et al., 1995, Clark et al., 1996, Kirschfink, 1997). The recruitment of neutrophils into the injured brain significantly contributes to neural damage by the release of oxygen radical metabolites and hydrolytic enzymes. Because of these effects and the results of experimental models of cerebral ischemia and TBI, neutrophil accumulation in the brain is associated with increased secondary brain damage and poor neurologic outcome (Clark et al., 1996, Hudome et al., 1997, Stahel et al., 1998).
The complement (C) system consists of at least 30 proteins in fluid-phase and membrane-bound form which serve as other crucial mediators of inflammation (Rother and Till, 1988, Frank and Fries, 1991, Bellander et al., 1996, Kirschfink, 1997). Together, these proteins act in a cascade reaction as part of the innate immune response following infection or injury. Because C is a key player in the initial inflammatory response, it therefore has a role in mediating tissue destruction. Once the cascade is activated, proinflammatory fragments like the anaphylatoxins C3a and C5a are produced, as well as the membrane attack complex (MAC) C5b–C9. These peptide products mediate many of the biological effects associated with C, such as neutrophil chemotaxis, increased vascular permeability, and phagocyte degranulation; all of which can enhance tissue destruction (Kirschfink, 1997).
Studies have shown that C activation in the central nervous system (CNS) can lead to increased neurodegeneration in disorders such as Alzheimer’s disease and multiple sclerosis (Spiegel et al., 1997). In 1987, Becker et al. showed that C activation was increased in the sera of patients who had sustained head trauma, and that increased activation correlated with severity of brain damage. More recently, both C3 and factor B were found to be elevated in the cerebrospinal fluid (CSF) of head-injured patients (Kossmann et al., 1997). Other experiments have shown that rats treated with soluble complement receptor type-1 (sCR-1) before trauma can significantly reduce neutrophil accumulation in the brain post-injury, suggesting an important role for C in the pathologic effects of brain trauma (Kaczorowski et al., 1995).
The lateral fluid-percussion (FP) model of traumatic brain injury in rats is well-characterized and in routine use (McIntosh et al., 1989, Gennarelli, 1994). Injury is caused by placing a small craniotomy over the left parietal cortex followed by a rapid epidural injection of saline into the closed cavity. This produces an immediate displacement and deformation of the brain (McIntosh et al., 1989, Hicks et al., 1997). The FP model produces several clinically important features of TBI (McIntosh et al., 1989, Smith et al., 1991, Gennarelli, 1994), including: (1) vascular damage, followed by disruption of the blood brain barrier and extravasation of serum components into the brain (Cortez et al., 1989, Dixon and Hayes, 1995, Kossmann et al., 1997), (2) neuronal damage, as evidenced by a loss of the neuron-specific cytoskeletal protein called microtubule-associated protein 2 (MAP2) (Hicks et al., 1995, Lewen et al., 1996), (3) reduced cardiac output and hypoperfusion in kidneys, spleen and liver (Yuan et al., 1990), and (4) leukocyte infiltration into the brain, including neutrophils and macrophages (Soares et al., 1995, Grady et al., 1999).
We proposed that this post-traumatic neutrophil influx may be associated with the accumulation of the complement component C3. To examine this hypothesis, we first wanted to determine the temporal and anatomical features of neutrophil infiltration after injury. For this purpose, we performed manual neutrophil counts and developed an improved myeloperoxidase (MPO) microassay that rapidly detects and quantifies any neutrophil accumulation in minute quantities of brain following FP injury. Myeloperoxidase is a hydrogen peroxide oxidoreductase found in the azurophilic granules of neutrophils that plays an important role in host defense against microorganisms (Bretz and Baggilioni, 1974, Klebanoff, 1975). Measurement of MPO activity as an enzyme marker to detect neutrophil accumulation in cutaneous inflammation was developed by Bradley et al. (1982). The use and sensitivity of this assay have dramatically increased with the optimization of assay reagents, temperature controls, and pH conditions (Suzuki et al., 1983, Schierwagen et al., 1990). Our improved MPO assay takes into account all of these optimum modifications and allows for successful MPO estimation even with minute quantities of tissue samples.
Our next step was to determine whether C3 accumulation is associated with neutrophil infiltration at the same anatomical sites and time points. The presence of C3 in the brain was detected by standard immunocytochemistry techniques (described in materials and methods). Western blots were performed to show that C3 was not only present in the brain, but also fragmented. Using the improved MPO microassay and C3 immunostaining procedures in this study, we have determined that there is an increased accumulation of both neutrophils and C3 in cortical tissue, as well as increased C3 in the ipsilateral hippocampus from FP-injured animals. The western analyses confirmed that the accumulated C3 was present as activated fragments. These results suggest that FP brain injury leads to local tissue damage that may be mediated by these precursors of proinflammatory effectors.
Section snippets
Lateral fluid-percussion injury
The fluid-percussion device (Biomedical Engineering Facility, Virginia Commonwealth University) consists of a plexiglass cylindrical saline-filled reservoir, 60 cm long and 4.5 cm in diameter, bounded at one end by a plexiglass, cork-covered piston mounted on O-rings. The opposite end of the reservoir is fitted with a 2 cm long metal housing on which a transducer is mounted and connected to a 5 mm tube that terminates with a male Leur-Loc fitting. At the time of injury, the tube is connected to
Results
The results using human MPO standards showed a linear regression curve with repeated reproducibility in the range from 0.025 U/ml to 1.0 U/ml (Fig. 1A). The concentrations above and below this did not fall within the linear range and were not used for further assays. Rabbit pulmonary MPO activity was measured in order to compare results obtained from the macroassay (tubes) and the microassay (plate). The results were comparable for all the samples in both tubes and plates, indicating that
Discussion
Our first goal in characterizing the proinflammatory aspects of the FP model for traumatic brain injury was to develop a more rapid and sensitive assay for quantifying tissue MPO levels. Although the MPO method described by Bradley et al. (1982) was an improvement, we still needed a faster method that would require only minute quantities of tissue. Our improved MPO microassay reported here fulfills that need, using one-tenth the sample volume. Our comparison data clearly demonstrates that there
Acknowledgements
The authors thank Chunying Li and David N. Reynolds for excellent technical assistance. This work was supported by Kentucky Spinal Cord and Head Injury Research Trust. In this study, we carefully adhered to the animal welfare guidelines set forth by the Institutional Animal Care and Use Committee at the University of Kentucky and the Guide for the Care and Use of Laboratory Animals from the U.S. Department of Health and Human Services.
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