Background
Traumatic brain injury (TBI) remains a major health burden in both developed and developing countries. TBI consists of two temporal pathological phases spanning the initial traumatic impact and a multitude of secondary cascades, resulting in progressive tissue degeneration and neurological impairment [
1‐
3]. The pathological consequences of TBI can be variable and largely depend on the presentation of injury as either focal or diffuse, or a combination of both. Diffuse brain injury may result from rotational forces and/or acceleration/deceleration of the head during a traumatic impact, often leading to diffuse axonal injury. Although difficult to diagnose due to the absence of lesions or overt pathology [
4,
5], diffuse axonal injury is a common presentation, accounting for up to 70% of all TBI cases [
6]. The pathology of diffuse axonal injury develops over a delayed time course, and is frequently aggravated by the occurrence of subsequent insults, which are known to worsen morbidity and mortality in TBI patients [
7]. Epidemiological studies have revealed that up to 44% of severe head trauma patients experience brain hypoxia, which has been associated with adverse neurological outcomes [
8‐
13]. Hypoxia can be initiated by TBI-induced cerebral hypoperfusion, apnoea and hypoventilation mostly related to brainstem injury [
14‐
16]. In addition, systemic hypoxia can be caused by extracranial injuries often co-existing with head trauma such as obstructed airways, lung puncture and excessive blood loss [
9,
17]. Despite these clinical observations, the exact mechanisms leading to the exacerbation of brain damage concomitant to posttraumatic hypoxia remain to be elucidated.
One putative sequel of TBI in contributing to secondary tissue damage is the activation of cellular and humoral neuroinflammation. This response is characterised by the accumulation of inflammatory cells in the injured area, as well as the release of pro- and anti-inflammatory cytokines, which may either promote the repair of injured tissue, or cause additional damage [
18]. The activation of inflammatory cascades in human and rodent TBI have previously been reported [
19‐
21]. In severe TBI patients, ourselves and others have demonstrated a robust longitudinal increase of multiple cytokines and chemokines in cerebrospinal fluid (CSF) [
22‐
27]. More recently, these findings have been corroborated with the upregulation of TNF, IL-1β, IL-6, IFN-γ protein and gene expression in post-mortem human brain tissue after acute TBI [
28]. Animal models of brain hypoxia or trauma can independently activate acute expression of cytokines IL-1β, IL-6 and TNF [
29‐
31]. Furthermore, in models of focal TBI, additional post-traumatic hypoxia was shown to worsen brain tissue damage [
32‐
34], cerebral edema [
35], and exacerbate sensorimotor, behavioural and cognitive impairment [
32,
34,
36‐
38]. The detrimental role of neuroinflammation can be elicited by its ability to induce the production of excitotoxic substances including reactive oxygen and nitrogen radicals [
39‐
41] contributing to the development of brain edema [
42,
43], blood brain barrier (BBB) disruption [
44,
45], and apoptotic cell death [
43,
46‐
49]. However, almost all the studies on post-TBI hypoxia used focal brain injury models, while epidemiological data on large patient populations reported that the majority of TBI patients present with diffuse brain injury leading to worse neurological outcome especially if associated with hypoxia [
6]. The few studies by us and others examining the effect of post-traumatic hypoxia after diffuse traumatic axonal injury (TAI; the experimental counterpart of human diffuse axonal injury) have demonstrated enhanced neurological deficits [
34,
38], exacerbated edema and cerebral blood flow, and diminished vascular reactivity [
50‐
54]. In a recent study using the Marmarou rat model of diffuse TAI with additional post-trauma systemic hypoxia, we demonstrated a greater axonal damage in the corpus callosum and brainstem co-localising with a robust macrophage infiltration and enhanced astrogliosis, when compared with TAI animals without hypoxia [
54‐
56]. Therefore, using this model of TAI, we aimed to further investigate whether post-traumatic hypoxia also aggravates behavioural and sensorimotor function, cerebral edema, enlargement of lateral ventricles, production of inflammatory cytokines in the brain, and impairment in cerebral energy metabolism.
Methods
Induction of trauma
Animal experiments were conducted in accordance with the Code of Practice for the Care and Use of Animals for Scientific Purposes (National Health and Medical Research Council, Australia), and received approval from the institutional Animal Ethics Committee. Adult male Sprague-Dawley rats were housed under a 12-hour light/dark cycle with food and water
ad libitum. Rats aged 12-16 weeks and weighing 350-375 g on the day of surgery were subjected to TAI (n = 27), TAI followed by a 30-min systemic hypoxia (TAI+Hx; n = 27), hypoxia only (n = 27) or sham surgery (n = 27). Briefly, rats were anaesthetized in a mixture of 5% isoflurane in 22% O
2/78% N
2, intubated, and mechanically ventilated with a maintenance dose of 2-3% isoflurane in 22% O
2/78% N
2. A steel disc (10 mm in diameter and 3 mm thickness) was adhered to the skull between bregma and lambda suture lines using dental acrylic. Animals were briefly disconnected from the ventilator and moved onto a foam mattress (Type E polyurethane foam, Foam2Size, VA, USA) underneath a trauma device where a weight of 450 g was allowed to fall freely though a vertical tube from 2 m. Following the impact, animals were reconnected to the ventilator, and ventilated continuously for a further 30 min using an appropriate concentration of isoflurane (0.5-1%) in either hypoxic (12% O
2/88% N
2) or normoxic (22% O
2/78% N
2) gas mixture. Consistent with the literature [
32,
36] we have previously demonstrated that such systemic hypoxic conditions result in an sO
2 of 47 ± 4.3% and pO
2 of 48.5 ± 3.8 mmHg, and cause a significant hypotensive episode, with mean arterial blood pressure (MABP) dropping to 69.5 ± 29.5 midway through the insult (i.e. 15 min). The reduction of sO
2, pO
2, and MABP returned to sham values by 15 min following the conclusion of the hypoxic period [
55]. Consistent with the original description of this model by Foda et al. (1994) [
40], the intubation and ventilation of rats after injury resulted in a mortality rate of ~10% which was confirmed in our study. When the two insults were combined, there was no significant increase in mortality. Hypoxia-only and sham operated animals were surgically prepared as described for TAI rats with the exception of the traumatic impact, and ventilated with hypoxic or normoxic gas, respectively. Rats were housed in individual cages after surgery and placed on heat pads (37°C) for 24 h to maintain normal body temperature during the recovery period.
Microdialysis probe implantation
Following trauma, 5 rats from each of TAI, TAI+Hx, hypoxia-only and sham groups were inserted with microdialysis probes into the brain for measuring real-time metabolite changes. If the microdialysis probe was implanted soon after the completion of TAI, high severity of the injury together with the ongoing anesthesia would result in a higher mortality rate. Therefore, we allowed the animals to recover for a period of 4 h before implantation of the microdialysis probe. Rats were then anesthetized by isoflurane, intubated and mechanically ventilated as described above. The head of the animal was immobilized on a stereotactic frame with nose and ear bars (David Kopf Instruments, California, USA). The scalp was opened at the existing suture line, and a 1-mm burr hole was drilled into the skull using a small handheld drill at the coordinates of -4.52 mm to bregma and -2 mm lateral to the midline on left hemisphere. Care was taken not to damage the dura mater. Two shallow holes were drilled posterior and anterior to the burr hole, and screws were inserted to provide anchor points for the microdialysis probe implantation. A guide cannula for CMA12 microdialysis probe was adjusted to 3 mm in length, inserted into the brain and secured in place by using dental cement (Dentsply, PA, USA) to cover both the guide cannula and the anchor screws. Once the dental cement solidified, the microdialysis probe (CMA12, 100 kDa cutoff, CMA Microdialysis, Solna, Sweden) was inserted into the guide tube to a suitable length allowing the semi-permeable membrane exposure outside of the guide tube for direct contact with the brain tissue. The microdialysis probe was immobilized by applying additional dental cement over the probe and guide cannula. At surgery completion, animals were allowed to recover in a microdialysis experimental system (CAM 120, CMA Microdialysis) which consists of a balanced arm with dual channel swivel allowing free movement of the animal and continuous collection of microdialysis samples. The microdialysis probe was perfused at 1 μl/min using artificial cerebrospinal fluid (aCSF, CMA Microdialysis). The effluent was collected as accumulative sample over 3 h (i.e. 180 μl/sample) using an automated refrigerated microdialysis fraction collector (Harvard Apparatus, MA, USA). Samples were transferred to -80°C freezer every 12 h and stored until analysis. At the end of the experimental period, animals were killed and brains were perfusion fixed to identify the location of the microdialysis probe in the cortex. Only the animals with the probe tip in the designated location were included for analysis.
Assessment of sensorimotor functions
Rats were treated in each group as described above and used for assessment of sensorimotor deficit by the Rotarod test, beam balancing and walking test, and adhesive tape removal from forepaws test (n = 10 per group). Animals were trained for these tasks every second day starting 1 week before surgery. These sensorimotor tests were performed daily after TAI for a week, then on every second day until 14 days. The Rotarod allows assessment of movement coordination and function including motor, sensory and balancing skills. Rats were placed on a rotating cylinder made of 18 rods (1 mm diameter) (Ratek, VIC, Australia). The rotational speed of the device was increased in increments of 3 rpm/5 sec, from 0 to 30 revolutions per minute (rpm). The maximal speed at which the rat was unable to match and failed to stay on the device was recorded. Body balancing and walking was assessed using a beam-walking test, in which rats were placed in the middle of a 2-meter long, 2-cm wide beam suspended 60 cm above the ground between 2 platforms. Rats were scored as: [
1] normal walking for at least 1 meter on the beam; [
2] crawling on the beam for at least 1 m with abdomen touching the beam; [
3] ability to stay on the beam but failure to move; and [
4] inability to balance on the beam. Sensory and fine motor function was assessed by the ability to remove adhesive tapes (5 × 10 mm; masking tape, Norton Tapes, NSW, Australia) placed on the back of each forepaw. The number of tapes removed (0, 1 or 2) and the latency for each tape removal were recorded within a 2-minute period.
Open field test
This test evaluates the animal's normal exploratory behavior. Rats were placed in an empty arena (70 × 70 × 60 cm, W×L×H) within an enclosed environment and low lighting. The movement of the rats was recorded for 5 min by a camera, and the distance walked was calculated using a custom made automated movement-tracking program (Dr Alan Zhang, Department of Electrical Engineering, The University of Melbourne).
Brain edema measurement
Rats with TAI, TAI+Hx, hypoxia or sham surgery were generated for assessment of brain edema. The wet-dry weight method was used for determining the water content of the brain at 2, 24, 48, 72, and 96 h after treatment (n = 6 per timepoint per group). Briefly, the left hemisphere was separated from the rest of brain tissue, weighed on a precision microbalance (Ohaus Adventurer Analytical Balance Bradford, MA, USA), and dried in an oven at 100°C for 24 h. The dry tissue was weighed again, and cortical water content was calculated as ([wet tissue weight - dry tissue weight]/wet tissue weight) × 100.
Measurement of ventricle size
A cohort of rats for each experimental group was treated as described above and killed at 1 or 7 days after injury (n = 6 per group per timepoint). Brains were perfusion fixed using 4% paraformaldehyde and embedded in paraffin wax. Brain tissue blocks were cut into 10 μm sections at the level of +1 mm relative to the bregma and collected onto glass slides. Sections were dewaxed, rehydrated, stained using hemotoxylin and eosin, and visualized under a light microscope (Olympus BX50). Multiple photographs were taken under 200× magnification to cover the entire sections. Image analysis software (ImageJ, NIH, USA) was used to align images taken from the same brain section to reconstruct a full section view. The whole brain area and the area of the ventricle were measured using ImageJ, with the area of the ventricle expressed as the percentage of total brain area.
Cytokine measurements
The right hemisphere from each animal of edema study was dissected, the cortex isolated, and stored at -80°C until use. The cortex was homogenised in an extraction solution containing Tris-HCl (50 mmol/L, pH 7.2), NaCl (150 mmol/L), 1% Triton X-100, and 1 μg/mL protease inhibitor cocktail (Complete tablet; Roche Diagnostics, Basel, Switzerland) and agitated for 90 min at 4°C. Tissue homogenates were centrifuged at 2000 rpm for 10 min, and the supernatants stored at -80°C until use. The concentration of 6 cytokines (IL-1β, IL-2, IL-4, IL-6, IL-10, TNF) in the brain cortex homogenates was determined by multiplex assay as previously used in our group [
57] (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, colored beads conjugated with cytokine antibodies were loaded into wells of 96-well filter plate. Following washing, the standards, quality controls and samples were added into the wells and incubated overnight at 4°C on a shaking platform. The wells were washed by filtration, and subsequently a solution with a mixture of biotinylated antibodies against each cytokine was added and incubated for 1 h at room temperature. Following the removal of excessive detection antibodies, streptavidin-phycoerythrin was added. Cytokine concentration was measured using multiplex assay reader (Bio-Rad Laboratories) and calculated against the standard curve. Total protein concentration was determined in each sample using the Bradford Assay (Bio-Rad Laboratories).
Analysis of microdialysis samples
The microdialysis samples (180 μl/sample, n = 5 per group) were freeze dried and suspended in small volume of ddH2O to increase the concentration of solutes. The samples were then analysed for glucose, lactate and glutamate using conventional enzymatic techniques performed in the ISCUS Analyser (CMA Microdialysis). Due to a substantial time delay between sample collection and analysis, pyruvate was not measured as it is known to be unstable after storage time of more than 3 months (CMA Microdialysis). The concentrations of glucose, lactate and glutamate in each sample were calculated to the original concentration according to the sample volume before and after the freeze-drying procedure.
Data analysis
Sensorimotor function assessment, cytokine concentration, brain metabolites and brain edema results were analysed using two-way repeated measures ANOVA. The open field test and ventricular size measurement were analysed by 1-way ANOVA. Data were presented as mean ± standard error of the mean. Data were considered as significant where p < 0.05.
Discussion
Cerebral hypoxia, along with hypotension, is one of the most critical factors worsening secondary brain damage after TBI, and particularly following diffuse TBI [
6,
13]. Despite this clinical relevance, the underlying mechanisms by which hypoxia aggravates neurological outcome following TBI have not been studied adequately.
Using focal or mixed focal-diffuse models, systemic hypoxia following TBI in rats exacerbates neurological deficit [
32,
37] and increases the lesion size, neuronal death [
33,
34,
37,
64] and brain edema, while reducing cerebral blood flow [
35,
51]. However, the role of post-traumatic hypoxia elicited after diffuse brain injury has rarely been addressed. Therefore, we explored the impact of hypoxia using a model of diffuse TAI [
40,
65,
66] followed by a 30-min hypoxic ventilation. Using this combinatorial insult model, we previously reported enhanced axonal damage and macrophage infiltration within the corpus callosum and the brain stem [
55]. Thus, in this follow-up study we further investigated changes in neurological outcome, brain edema, ventricle enlargement, cerebral cytokines, and energy metabolism.
We found that in comparison to TAI alone, an additional hypoxic insult enhanced sensorimotor deficits on the Rotarod, beam walk and tape removal tests, reduced spontaneous exploratory behavior, and delayed recovery. These data closely relate to clinical studies on TBI patients showing that post-traumatic hypoxia worsens neurological outcome and prolongs the recovery period [
7,
8,
67]. The behavioural data in this model of TAI are consistent with similar deficits shown at day 1 in previous studies using diffuse or focal TBI models in combination with hypoxia [
32,
34,
36‐
38,
68]. However, in extension of this early work, our results show that an additional hypoxic insult has a detrimental effect on behaviour, inflammatory and metabolic outcomes for an extended period of time.
Brain swelling is a major contributor for the development of secondary ischemia causing raised ICP and decreased cerebral perfusion pressure [
69]. Enlargement of the brain due to edema [
70] and/or obstruction of CSF flow [
71] is a common event in severe TBI patients and a frequent cause of death. Cytotoxic edema results from excessive accumulation of ion and water within the cell, while vasogenic edema is caused by increased vascular permeability and subsequent fluid extravasation into the parenchyma. Here, we demonstrated that at 2 h after TAI, brain water content was similar to sham animals, but it increased to a peak between 24 and 48 h, and remained elevated until 72 h. Although hypoxia following TAI exacerbated sensorimotor deficit, it did not further increase cerebral edema when compared with TAI only animals, corroborating previous observations using diffuse-weighted imaging [
35]. Interestingly, using MRI, others demonstrated that acute brain swelling after TAI (both with and without hypoxia), as early as 60 min post-injury, was associated with increased extracellular fluid and BBB dysfunction, indicative of vasogenic edema [
72‐
75]. This early brain swelling was transient, with values quickly returning to sham levels [
53,
58,
75]. Since the earliest timepoint examined in our study was 2 h, it is likely that we missed this initial peak in edema, as no differences were detected between TAI, TAI+Hx and sham rats later on. However, other studies have also demonstrated that a modest, widespread second edematous response occurs at 24 h after TAI despite the intact BBB, which suggests ongoing cytotoxic edema [
58,
75]. Our results are consistent with this modest yet significant increase of edema at 24 h, which was maintained until 48 h. It is possible that the peak in brain water content observed at 24 h in both the TAI and TAI+Hx rats (approximately 79.3%) reflects a sort of saturation level, with the brain unable to tolerate any further water accumulation. Other studies also demonstrated peak edema of similar degree after TBI [
59,
76,
77].
An interesting observation was the enlargement of the lateral ventricles after TAI, and even greater following TAI+Hx. Recent clinical neuroimaging studies have shown correlations between ventricular enlargement and long-term neurological impairment [
78‐
80]. The prognostic value of ventricular dilatation had high sensitivity and specificity for the prediction of cognitive outcome [
80‐
83]. In this study, we showed that the lateral ventricles are markedly enlarged at 1 day post-injury after TAI and even larger in TAI+Hx animals, when compared to sham or rats with isolated hypoxia. Although we did not examine the mechanism leading to ventricular enlargement after TAI, imaging studies on TBI patients suggested that white matter degeneration around the lateral ventricle may be a contributing factor [
84]. However, since ventricular enlargement in TAI rats was an early and transient effect, it could be most likely attributed to the onset of post-traumatic hydrocephalus, caused by impaired CSF circulation due to edema compressing the aqueduct of sylvius.
Neuroinflammation has been extensively investigated in hypoxia-ischemia and TBI in both humans and animal models [
85] and all these studies have reported a robust elevation of cytokines in the central nervous system [
19,
28,
86‐
89]. More relevant for this study, our preliminary data on severe TBI patients with additional hypoxic insult have shown enhanced and prolonged production of cytokines in the CSF (Yan et al: Neuroinflammation and brain injury markers in TBI patients: Differences in focal and diffuse brain damage, and normoxic or hypoxic status on neurological outcome; manuscript in preparation). Consistently, here we demonstrated exacerbated production of IL-6, IL-1β, and TNF in the brains after TAI with additional hypoxia.
IL-1β is a key mediator of the inflammatory response, which exacerbates neuronal injury and induces BBB dysfunction by stimulating matrix metalloproteinases [
90]. IL-1β mRNA is upregulated within minutes after TBI, and increased protein levels are detectable within an hour after TBI [
21,
91‐
93]. In this study, IL-1β increased early after TAI alone, peaking at 2 h. Post-TAI hypoxia significantly enhanced IL-1β concentration at 2 h compared to TAI-only rats. In addition, whilst the elevation of IL-1β in TAI-only rats appeared to be transient, in TAI+Hx rats IL-1β was still significantly elevated at 24 h, suggesting that the addition of hypoxia prolongs neuroinflammation.
The neurotoxic effects of IL-1β are synergistically enhanced in the presence of TNF [
94], as both share many of the same physiologic effects. However, the role of TNF following TBI is controversial, neuronal toxicity of TNF has been demonstrated with local TNF administration inducing breakdown down of the BBB and increased leukocyte recruitment [
95‐
98]. Clinically, high levels of TNF in the CSF of brain-injured patients correlated with BBB dysfunction [
99]. TNF inhibition also reduced cerebral ischemia/reperfusion injury [
100], decreased TBI induced neuronal damage [
101], and ameliorated BBB dysfunction after closed head injury [
102]. However, studies on TNF deficient mice demonstrated an early functional improvement between 24-48 h after TBI, but failed to produce further amelioration at 4 weeks [
103]. Taken together, these studies suggest that TNF may be deleterious in the acute phase post-injury, but beneficial for long-term recovery. In accordance with Kamm et al. [
93], no changes in TNF levels were detected in rats subjected to TAI alone, whereas the combination of TAI and hypoxia elicited a significant early increase in TNF at 2 h post-injury, lasting up to 72 h post-injury. These early enhancement in the TAI+Hx rats possibly reflects a more severe brain damage in this combined insult model.
Similar to IL-1β and TNF, at 24 h IL-6 was significantly higher in TAI+Hx rats compared to TAI alone. IL-1β is an early mediator inducing the production of IL-6 at both mRNA and protein levels [
21]. IL-6 displays pleiotropic functions with both deleterious and beneficial effects in the injured brain [
104‐
106]. Using the mild severity (250 g/2 m) of the Marmarou model, we showed that IL-6 increased in rat CSF within 24 h and IL-6 protein and mRNA was found expressed on neurons [
95]. Studies of IL-6 gene-deficient mice have provided more information in regards to the protective function of IL-6, by having a compromised immune response, increased oxidative stress and neurodegeneration [
107]. In this study, we demonstrate significantly heightened IL-6 levels in the TAI+Hx rats at 24 h, which remained elevated above TAI levels until 96 h. Altogether, the increased acute production of IL-1β and TNF may be associated with disruption of BBB integrity and consequently formation of cerebral edema, while late elevation of IL-6 may trigger repair mechanisms [
24,
99].
We also investigated changes in energy metabolism in this combinatorial insult model. Due to the nature of the impact acceleration injury, it is impractical to implant a microdialysis probe prior to injury without compromising the integrity of the trauma. It is also difficult to implant the probe directly after trauma as it resulted in higher mortality rate. Carré and colleagues implanted the probe 2 weeks prior to injury, but without success [
108]. We therefore allowed the rats to recover for 4 hours after TAI before implanting the microdialysis probe. In accordance with others [
63], our study has shown that in sham rats energy metabolism is altered during the first 24 h following microdialysis probe implantation, therefore we chose to examine only the data from 20 h onwards to reduce the "probe effect".
At 21 h, the glucose values for TAI+Hx rats were substantially lower compared to TAI or sham rats, and dropped to extremely low levels from 57 h onwards. These low levels of cerebral glucose could be the result of low glucose availability and/or hyperglycolysis in the acute post-injury phase. Hyperglycolysis has previously been shown as common early event following neurotrauma both experimentally and in the clinic [
109,
110]. It is often followed by a prolonged period of metabolic depression beginning as early as 6 h post-injury, remaining for as long as 5 days [
111,
112], a phenomenon which has also been demonstrated in the present study. Interestingly, rats subjected to TAI experienced only a brief period of glucose depletion between 39 h and 57 h, at which time glucose levels returned to sham values for the remaining duration of monitoring. It is possible that the additional hypoxic insult depleted available glucose stores in the TAI+Hx animals, and thus a prolonged compensatory period of anaerobic respiration occurred to provide essential ATP and generate lactate as by-product. Our experiments have demonstrated that this is a protracted process, lasting for 51 h after TAI. Lactate may be utilized by the brain during periods of increased brain energy requirements in which ATP and glucose stores are exhausted, such as following TBI [
113,
114]. In a situation of prolonged glucose depletion, high concentrations of lactate and high-level energy usage for neuronal repair or alternative metabolic pathways may further reduce the ATP reserves, with a subsequent mismatch between glucose transport, uptake and ATP production [
115,
116]. This may explain the further drop in glucose concentrations at 57 h post TAI, in that the restoration of aerobic metabolism decreases lactate concentration but further reduces glucose. Post-traumatic impairment in energy metabolism is a major contributor to cytotoxic edema, and interestingly, the period of elevated lactate in the TAI+Hx rats between 21 h and 57 h overlaps with the peak of increased brain water content. As edema begins to reside, lactate levels in these rats return to sham values. This prolonged period of metabolic crisis also extends to glutamate production, which was depressed below sham levels for TAI, and particularly TAI+Hx rats, for the duration of the monitoring by microdialysis.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
EBY designed the study, performed all animal work and microdialysis probe implantation, performed cytokine measurements, drafted the manuscript, and performed statistical analysis. SCH assisted with animal work, performed sensorimotor experiments, carried out the histology and ventricle measurements, performed statistical analysis, and drafted the manuscript. BMB carried out microdialysis sample measurements and assisted with manuscript preparation. DAA assisted with animal work, carried out sensorimotor and open field exploration experiments, and performed edema experiments. CMK conceived of the study and oversaw its design and coordination, and drafted the manuscript. All authors have read and approved the final manuscript.