Traumatic brain injury (TBI) is present in two-thirds of patients with multiple injuries (1-3). Data from the German Trauma Registry data base (DGU Traumaregister 1993-2002; n = 9646) confirmed that 57.5% of multiply injured patients suffer from a relevant TBI (Abbreviated Injury Scale ≥3); in one-third, TBI is combined with injuries of the extremities. Despite these clinical observations, experimental research to date has focused primarily on “single-hit” models to uncover principle mechanisms involved in trauma. Inflammatory responses known for their crucial role in determining the patient's fate (4-6) have been extensively but almost exclusively studied in the isolated human and/or experimental setting. Thus, little is known about interactive effects among central and peripheral injuries when both occur simultaneously in the sense of a combined neurotrauma (CNT), although there is increasing clinical evidence for possible interactions between central and peripheral injuries (7-9) affecting outcome (2, 10, 11).
One reason why CNT has been neglected as a research target so far might have been the absence of clinically relevant experimental models (12, 13). The first studies were conducted on TBI combined with hemorrhage, but focused primarily on resuscitation or ventilatory protocols (14, 15). Wichmann et al. (16) studied immunological responses after combined bone fracture, soft-tissue damage, and hemorrhage, but not TBI. To date, no experimental model has been described and thus no experimental data exists on the pathophysiological sequelae after combined trauma involving TBI and bone fractures. Results from such investigations may have a profound impact on the prevention of secondary injury and may provide insight into the time window for therapeutic interventions, e.g., management and timing of fracture repair (17, 18), in the frequent clinical scenario of multiple trauma.
To meet the growing demand for clinically relevant but experimental “more-hit” models in trauma research, a new model of combined neurotrauma incorporating the standardized lateral fluid percussion brain injury model/lateral fluid percussion (LFP) (19), together with the peripheral bone fracture model, i.e., tibia fracture (20), is introduced. In a first attempt to implement this model, we investigated whether it would reproduce some characteristic features known from human multiple trauma with respect to the post-traumatic inflammatory response as exemplified by circulating cytokine interleukin 6 (IL-6) levels (4-6) and production of callus at the fracture site. There are several reports that indicate that the production of callus and the rates of healing of fractures are increased by concomitant head injury (21-23), and only recently the possible involvement of IL-6 in altered fracture healing after head injury has been advocated (24).
MATERIALS AND METHODS
Overview of experimental animal groups
Male Sprague-Dawley (SD) rats (n = 100; 300-350 g) were commercially obtained from Harlan-Winkelmann (Borchen, Germany) and housed in individual cages for a minimum of 1 week before any procedure. After acclimatization, animals were randomly assigned to one of the following experimental groups: controls (n = 10), TBI only via moderate brain injury/LFP (n = 30), tibia fracture only (n = 30), and CNT, i.e. TBI only plus tibia fracture only, (n = 30). For induction of both traumatic impacts, TBI and tibia fracture, and further procedures, see below. Postinjury, animals were returned to their home cages with food and water continuously available. All animals were maintained at constant temperature (22°C) in a 12-h light/dark cycle, with lights on at 7:00 a.m. All surgical procedures were carried out under aseptic conditions. All experimental procedures conformed with the guidelines of the University of Cologne and the state's animal protection and ethics committee. All efforts were made to minimize animal discomfort and to reduce the total number of animals used.
The LFP brain injury model is one of the most widely used models of experimental TBI and has been described elsewhere (19). It is furthermore considered the one that most closely mimics postlesional events associated with TBI in humans (19, 25). In brief, animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), placed in a stereotactic frame, and the scalp and temporal muscle were reflected. A hollow female Luer-Lok fitting was rigidly fixed with dental cement to a 4.8-mm craniotomy centered between bregma and lambda and 2.5 mm lateral to the sagittal sinus, keeping the dura mater intact. The fluid percussion device consists of a Perspex cylinder filled with isotonic saline. One end of the cylinder is connected to a metal housing terminated with a male Luer-Lock fitting. Before trauma induction, the male Luer-Lok was connected to the female Luer-Lok anchored in the rat's skull, creating a closed system filled with isotonic saline in connection with the dura. Trauma was induced by the fall of a metal pendulum against the piston inducing a pulse of increased intracranial pressure of 21 to 23 ms in duration through the rapid injection of saline into the closed cranial cavity, thus resulting in a brief displacement and deformation of neural tissue. The pressure pulse was measured extracranially by a transducer (Gould Inc., Oxnard, CA) housed in the injury device and recorded on a computer oscilloscope emulation program (RC Electronics, Santa Barbara, CA). After injury at a moderate level (2.1 atm), the incision was closed with interrupted 4.0 silk sutures and the animals were placed in a heated cage to maintain body temperature for 1 h after surgery. All animals were monitored for at least 6 h postsurgery, and were then monitored daily.
A reproducible method for producing bone fractures, i.e., tibia fractures, was adopted from Bourque et al. (20). In brief, a custom-designed fracture apparatus was constructed that can be operated by a single person (see Fig. 4 later in the text). The dimensions of the apparatus are as follows: base, 30 cm long × 12 cm wide × 9 cm high. The base was constructed of Perspex. Attached to the base is a brass fulcrum and a brass lever. The tibia was placed across the fulcrum. The apparatus was operated by swinging the lever down over the fulcrum. Contact to the tibia was assured by two points on the lever located on either side of the fulcrum.
For the model of CNT, both models, the LFP brain injury model and the bone fracture model, were combined. Fractures were induced immediately after LFP under the same anesthesia as described above.
Neuromotor dysfunction: composite neuroscore (NS) test
Animals subjected to TBI only that survived >24 h postinjury and controls were tested for gross neuromotor dysfunction and recovery using a composite NS test (26, 27). The results obtained from this test correlate with injury severity (19, 28). In brief, animals were tested by an investigator blinded to the injury status of each animal. Scoring for each individual animal ranged from 0 (severely impaired) to 4 (normal strength and function) for each of the following modalities: (1) left and (2) right forelimb flexion during suspension by the tail; (3) left and (4) right hind limb flexion with the forelimbs remaining on a flat surface as the hind limbs are lifted up and down by the tail; (5) ability to resist lateral pulsion to the left and (6) right; (7) ability to stand on an inclined plane in the left, (8) right, and (9) vertical position. Inclined plane scoring (0-4) was determined by the animal's ability to stand at an angle up to 45 degrees (4 = 45°; 3 = 42.5°; 2 = 40°; 1 = 37.5°; 0 = <37.5°). The scores for 7, 8, and 9 were averaged, and a composite neurological motor score (0-28 points) was calculated for each animal from the summation of the individual test scores. Baseline composite NS was performed 24 h before injury, and neuromotor function was then evaluated at 24 h and 7 days postinjury.
Neuromotor dysfunction in fractured animals: modified composite NS test
To demonstrate presence or absence of neuromotor dysfunction in the two other experimental groups, i.e., CNT and fracture only, a modified composite NS was applied sparing those functions impaired by the fracture. In those animals, bilateral forelimb flexion (1 and 2) and uninjured hind limb flexion (3) was assessed at same time points as for animals subjected to TBI only (see above), and a reduced composite NS (0-12 points) was calculated for each animal.
Animals subjected to TBI only that survived 24 to 48 h postinjury were analyzed for TBI-induced histopathological changes. Animals were reanesthetized with pentobarbital (60 mg/kg, i.p.), transcardially perfused with 0.9% NaCl in distilled water followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and decapitated. The brains were rapidly removed from the cranial cavity and fixed in the same solution for another 4 to 6 days. Thereafter, the brains were cut into consecutive 25-μm sections in the coronal plane using a vibratome, mounted on slides, and stained with crysel-violet for light microscopy. To confirm that the fractures produced were complete and reproducible, conventional X-rays were taken in two planes from the fracture site at 24 h postinjury from animals subjected to tibia fracture only.
Blood sampling and IL-6 immunoassay
Blood samples were drawn before trauma (controls), at 30 min, and at 6 h, 24 h, 48 h, and 7 days after trauma under anesthetic conditions as described above via direct puncture of the left ventricle of the heart using EDTA as anticoagulant (EDTA/KE 7.5-mL monovettes [Sarstedt, Nümbrecht, Germany]). Thereafter, blood samples were centrifuged at 2000g within 30 min after collection and they were frozen at −40°C until further analysis. Plasma samples were analyzed using a commercial rat IL-6 immunoassay kit (Quantikine M rat IL-6 immunoassay; R&D Systems, Minneapolis, MN).
Callus formation at fracture sites
One subset of animals (n = 15; five animals each from groups TBI only, tibia fracture only, and CNT) was prepared for callus volume assessment 15 days postinjury. After the animals had been sacrificed as described above, the fractured tibias were prepared surgically, then dehydrated and defatted in acetone (−20°C) to allow uniform penetration of the resin. In this study, we used a methyl methacrylate-based resin (Technovit 9111 NEU; Kulzer, Wehrheim, Germany). After dehydration, infiltration of specimens was carried out under vacuum using graded resin/acetone series. Vacuum was applied to facilitate infiltration of the resin and to prevent air bubble formation. During the embedding procedure, special care was taken not to inhibit the polymerization process. The procedure yielded transparent and hard blocks containing fractured bones that were trimmed and cut equidistantly (500 μm) using an automatic diamond saw (Well; Präzionstechnik Sommer, Usingen, Germany). Semithin sections were mounted on slides and polished. Microradiographs were recorded, scanned, and analyzed for callus formation at fracture sites using Morphomet software. Callus volumes were calculated from the total area of all sections and the section thickness.
Data are expressed as means (±SD) after they were subjected to a two-way analysis of variance (ANOVA) followed by a post hoc Bonferroni t test. The distribution of IL-6 immunoassay data was analyzed to verify that it was normal. To compare IL-6 plasma levels among the three trauma groups, a general linear model with time being a categorical confounder was used. This approach avoids multiple comparisons at different time points and enables to test for interaction between groups and time. Post hoc analysis was performed with Tukey's test. A student's t test was used for exploratory pairwise comparisons among the different trauma groups and controls. Significance levels were set at P < 0.05.
Neuromotor dysfunction after TBI and CNT
The LFP brain injury model typically produced a neuromotor dysfunction contralateral to the injured site of the brain, resulting in weakness and a loss of reflexes and coordination. As expected, no differences were observed among intact animals before injury and controls with respect to their forelimb flexion, hind limb function, lateral pulsion, and baseline angles in the angle-board test (baseline; Fig. 1). However, at 24 h postinjury, a comparable level of severe neurological impairment was noted in all animals subjected to TBI only (P < 0.001). Although some degree of neuromotor recovery occurred during the first week postinjury, NS obtained at 7 days postinjury were still significantly lower compared with baseline scores (P < 0.001). A modified composite NS with a maximum score of 12 points sparing those functions impaired by the fracture was applied to animals subjected to CNT and fracture only. The neurocomponent of the model was also evident and reproduced in animals subjected to CNT (P < 0.001), whereas animals without brain impact, i.e., fracture only, generated maximum modified NS values across all time points studied (Fig. 2).
Histopathological observations after TBI
Figure 3 depicts histopathological findings from brain-injured animals at 24 to 48 h postinjury indicating a reproducible lesion located between Bregma −2.3 mm and −7.3 mm with several characteristic features. In detail, a cortical contusion ipsilateral to the injured site of the brain was evident in all animals, macroscopically and microscopically. Furthermore, a characteristic pattern of hemorrhage was visible in all animals typically occurring within the following anatomical regions: ipsilateral cortex, subcortical white matter/internal capsule, corpus callosum, hippocampus including fimbria hippocampi, as well as in the pons and the subarachnoid space between the thalamus and the hippocampus and the thalamus and the midbrain. In a few animals, intraparechymal petechiae were observed in the colliculus superius, the dentate gyrus, and the thalamus. Microscopic analysis revealed a mass shift toward the contralateral hemisphere as a consequence of the trauma-induced ipsilateral edema, as exemplified by additionally but selectively performed T2-weighted magnetic resonance imaging. Neuronal cell loss was evident in all animals predominantly in the adjacent cortex, as well as in the CA-2 and CA-3-regions of the ipsilateral hippocampus. However, other features associated with brain injury via LFP brain injury not shown here but confirmed by our group include micro- and astroglia activation (29) and metabolic alterations.
Conventional x-ray imaging 24 h postinjury from fracture sites indicated that the fractures produced were complete and localized in the middle of the bone shaft (Fig. 4).
Amplification of the IL-6 response by an additional bone fracture
Circulating IL-6 levels rose steeply compared with controls (Fig. 5) being statistically significant for all groups at all time points studied (P < 0.01 vs. controls by t test), except for animals with TBI only at 7 days after trauma. IL-6 levels in CNT were highest at all time points with a maximum peak of 387 pg/mL (mean) 6 h after trauma (P < 0.001 for both by t test). At 7 days after trauma, circulating IL-6 plasma levels in CNT were still elevated, whereas levels in other groups had continuously decreased after 48 h. The general linear model demonstrated that the type of injury (P < 0.001) and time (P = 0.022) were significantly associated with increased IL-6 levels, whereas the statistical interaction between the two variables was of borderline significance (P = 0.057). IL-6 plasma levels in CNT were considerably higher compared with TBI (P = 0.004) or fracture only (P = 0.006). After combined trauma, IL-6 levels were on average 93 pg/mL (95% confidence interval, 22-165) and 91 pg/mL (95% confidence interval, 25-158) higher than after isolated TBI or fracture. Accordingly, the two monotrauma groups showed very similar IL-6 plasma levels (P > 0.99).
Increased callus formation at fracture sites in CNT
Callus formation at fracture sites in CNT was significantly increased compared with animals with fracture only (P < 0.01). No callus formation was observed at bones harvested from animals exposed to TBI only (Fig. 6).
To meet the increasing demand to implement clinically relevant “more-hit” models in experimental trauma research, a new model of CNT consisting of a standardized TBI via LFP (19) and peripheral bone, i.e., tibia fracture (20) is presented. TBI, along with fractures of the extremities, is one of the most frequent injury pattern in clinical trauma (DGU Traumaregister 1993-2002; n = 9646). Epidemiological data demonstrates that 73% of severely injured patients suffer from TBI, followed by injuries of the extremities (70%); in one-third, TBI is combined with injuries of the extremities (1-3).
Our results indicate that both components of our model are robust and reproducible. Histomorphological analysis of the neurocomponent confirmed earlier reports (19, 30-32), thus emphasizing the value of the LFP model for human brain injury (25). X-ray imaging demonstrated that fractures were complete and routinely induced in the middle of the bone shaft as originally described (20). To confirm absence or presence of the neurocomponent in fractured animals that could not fully be assessed using the conventional composite NS due to hind limb impairment, a modified composite NS was successfully implemented.
Using this new model of CNT, two characteristic features known from human multiple injury involving TBI were successfully reproduced. First, while investigating the extent and the temporal pattern of the post-traumatic IL-6 response under different experimental conditions, we found that the type of injury and the time were significantly associated with increased circulating IL-6 levels. Plasma IL-6 levels after CNT clearly exceeded those observed after monotrauma. There appeared to be more than just an additive effect among injuries, as the peak maximum in the combined group at 6 h after trauma clearly exceeded the value of both monotrauma peak maxima taken together. Clinically, systemic IL-6 concentrations in plasma have been related to the trauma burden (4-6) and outcome (4, 33-35). Similarly, the results obtained from animals with isolated TBI and tibia fracture were consistent with earlier experimental and clinical findings (36).
Second, increased callus formation at fracture sites was significantly associated with animals that had sustained the additional neurocomponent of the model, i.e., CNT. This phenomenon has also been observed clinically as the production of callus and the rates of healing of fractures are increased by concomitant head injury (21, 22, 37). In addition, heterotopic ossification has frequently been reported from patients with traumatic paraplegia (21-23). Although yet unclear, neuronal or humoral mechanisms may play a role in the link between central injury and the formation of new bone at distant sites. Recently, IL-6 has been suggested to be involved in altered fracture healing after head injury (24). Similar to the present study, Beeton et al. (24) demonstrated that the circulating level of IL-6 is significantly raised in patients who have sustained head injury and fracture compared with uninjured control subjects and that the level remained high at later time points. Sera from head-injured patients thus containing increased IL-6 levels stimulated the proliferation of osteoblasts and the production of alkaline phosphatase in vitro (38, 39).
Our findings are in correspondence with earlier reports suggesting significant interactions between injuries at various body sites with reference to TBI. In the clinical scenario, mortality and outcome are significantly worsened when multiple injury is associated with TBI and vice versa (2, 7, 8, 10, 11). Mortality was not investigated in the present study, as a single bone fracture added to TBI was not considered to be sufficiently severe to affect this parameter in the model. Interactive effects between injuries may involve immunological dysfunction, as trauma-associated release of stress factors and brain cytokines may induce central nervous system-mediated immunodepression via lymphocytic and monocytic inactivation (40). A systemic inflammatory response syndrome may occur with a high risk for single- or multiorgan failure (SOF/MOF) and sepsis (33). For example, own clinical data revealed that SOF/MOF is more likely to develop in CNT versus monotrauma despite comparable overall severity (58% vs. 25%; E.A.M. Neugebauer, unpublished data from 328 patients). The presence of receptors for various neurotransmitters on immune cell surfaces and, vice versa, the presence of receptors for various cytokines on the surface of brain cells (41) underscores the potential for interaction of central and peripheral injuries, emphasizing the tight link between the cellular immune system and the central nervous system. An overview of current knowledge about bidirectional interactive effects between central and peripheral trauma on various levels and different acute versus chronic responses at least on the cytokine level has been published by our group (9).
The overall question to what extent interactions between injuries at various parts of the body occur, e.g., additive, synergistic, or counter-regulatory, remains as yet unclear. The choice of effective treatment, in particular with focus on secondary and therefore treatable damage, however, relies strongly upon mechanisms believed to be involved in multiple injury, including TBI. Identification of the neurochemical mediators and mechanisms underlying its etiology remains a central element for effective therapeutic intervention. With respect to the data presented here, it may be assumed that inflammatory acute-phase responses, characterized by elevated levels of various pro- and anti-inflammatory mediators, may play an even more deleterious role in CNT compared with monotrauma, as inflammatory IL-6 mean plasma levels in CNT exceeded those in isolated trauma more than 2-fold, not following a simple additive pattern. In addition, increased IL-6 levels in CNT, though not significant, persisted up to 7 days after trauma, suggesting a more prolonged response versus monotrauma. Because inflammatory cytokine IL-6 concentrations in plasma after trauma correlate with the amount of trauma burden, as confirmed in the present study, and outcome (4, 34), this may contribute to the increased susceptibility to early sepsis and single/multiorgan failure associated with whole body inflammatory response in clinical CNT (4).
Regardless of these first insights, the need for clinically relevant experimental models to precisely determine the complex pathophysiological events occurring with CNT remains undisputed (12). Here, a new and innovative model is introduced that mimics a variety of clinically relevant features known from human multiple injury including TBI, thus offering a unique experimental approach for further investigation of the complex pathophysiological, biochemical, and neuroimmunological mechanisms involved. We hope that clinically relevant experimental models, as introduced here, will be used in the future for clarification. Further studies are currently underway to more precisely characterize the immunological consequences produced by the model.
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Combined neurotrauma; fluid percussion; tibia fracture; inflammatory response; interleukin 6; callus formation; animal model