Post-traumatic hypoxia is associated with prolonged cerebral cytokine production, higher serum biomarker levels, and poor outcome in patients with severe traumatic brain injury.

Secondary hypoxia is a known contributor to adverse outcomes in patients with traumatic brain injury (TBI). Based on the evidence that hypoxia and TBI in isolation induce neuroinflammation, we investigated whether TBI combined with hypoxia enhances cerebral cytokine production. We also explored whether increased concentrations of injury biomarkers discriminate between hypoxic (Hx) and normoxic (Nx) patients, correlate to worse outcome, and depend on blood-brain barrier (BBB) dysfunction. Forty-two TBI patients with Glasgow Coma Scale ≤8 were recruited. Cerebrospinal fluid (CSF) and serum were collected over 6 days. Patients were divided into Hx (n=22) and Nx (n=20) groups. Eight cytokines were measured in the CSF; albumin, S100, myelin basic protein (MBP) and neuronal specific enolase (NSE) were quantified in serum. CSF/serum albumin quotient was calculated for BBB function. Glasgow Outcome Scale Extended (GOSE) was assessed at 6 months post-TBI. Production of granulocye macrophage-colony stimulating factor (GM-CSF) was higher, and profiles of GM-CSF, interferon (IFN)-γ and, to a lesser extent, tumor necrosis factor (TNF), were prolonged in the CSF of Hx but not Nx patients at 4-5 days post-TBI. Interleukin (IL)-2, IL-4, IL-6, and IL-10 increased similarly in both Hx and Nx groups. S100, MBP, and NSE were significantly higher in Hx patients with unfavorable outcome. Among these three biomarkers, S100 showed the strongest correlations to GOSE after TBI-Hx. Elevated CSF/serum albumin quotients lasted for 5 days post-TBI and displayed similar profiles in Hx and Nx patients. We demonstrate for the first time that post-TBI hypoxia is associated with prolonged neuroinflammation, amplified extravasation of biomarkers, and poor outcome. S100 and MBP could be implemented to track the occurrence of post-TBI hypoxia, and prompt adequate treatment.

Post-traumatic hypoxia exacerbates neurological deficit, neuroinflammation and cerebral metabolism in rats with diffuse traumatic brain injury.

BACKGROUND: The combination of diffuse brain injury with a hypoxic insult is associated with poor outcomes in patients with traumatic brain injury. In this study, we investigated the impact of post-traumatic hypoxia in amplifying secondary brain damage using a rat model of diffuse traumatic axonal injury (TAI). Rats were examined for behavioral and sensorimotor deficits, increased brain production of inflammatory cytokines, formation of cerebral edema, changes in brain metabolism and enlargement of the lateral ventricles. METHODS: Adult male Sprague-Dawley rats were subjected to diffuse TAI using the Marmarou impact-acceleration model. Subsequently, rats underwent a 30-minute period of hypoxic (12% O2/88% N2) or normoxic (22% O2/78% N2) ventilation. Hypoxia-only and sham surgery groups (without TAI) received 30 minutes of hypoxic or normoxic ventilation, respectively. The parameters examined included: 1) behavioural and sensorimotor deficit using the Rotarod, beam walk and adhesive tape removal tests, and voluntary open field exploration behavior; 2) formation of cerebral edema by the wet-dry tissue weight ratio method; 3) enlargement of the lateral ventricles; 4) production of inflammatory cytokines; and 5) real-time brain metabolite changes as assessed by microdialysis technique. RESULTS: TAI rats showed significant deficits in sensorimotor function, and developed substantial edema and ventricular enlargement when compared to shams. The additional hypoxic insult significantly exacerbated behavioural deficits and the cortical production of the pro-inflammatory cytokines IL-6, IL-1ß and TNF but did not further enhance edema. TAI and particularly TAI+Hx rats experienced a substantial metabolic depression with respect to glucose, lactate, and glutamate levels. CONCLUSION: Altogether, aggravated behavioural deficits observed in rats with diffuse TAI combined with hypoxia may be induced by enhanced neuroinflammation, and a prolonged period of metabolic dysfunction.

Neurogenesis and glial proliferation are stimulated following diffuse traumatic brain injury in adult rats.

Although increased neurogenesis has been described in rodent models of focal traumatic brain injury (TBI), the neurogenic response occurring after diffuse TBI uncomplicated by focal injury has not been examined to date, despite the pervasiveness of this distinct type of brain injury in the TBI patient population. Here we characterize multiple stages of neurogenesis following a traumatic axonal injury (TAI) model of diffuse TBI as well as the proliferative response of glial cells. TAI was induced in adult rats using an impact-acceleration model, and 5-bromo-2'-deoxyuridine (BrdU) was administered on days 1-4 posttrauma or sham operation to label mitotic cells. Using immunohistochemistry for BrdU combined with phenotype-specific markers, we found that proliferation was increased following TAI in the subventricular zone of the lateral ventricles and in the hippocampal subgranular zone, although the ultimate production of new dentate granule neurons at 8 weeks was not significantly enhanced. Also, abundant proliferating and reactive astrocytes, microglia, and polydendrocytes were detected throughout the brain following TAI, indicating that a robust glial response occurs in this model, although very few new cells in the nonneurogenic brain regions became mature neurons. We conclude that diffuse brain injury stimulates early stages of a neurogenic response similar to that described for models of focal TBI.

Post-traumatic hypoxia exacerbates brain tissue damage: analysis of axonal injury and glial responses.

Traumatic brain injury (TBI) resulting in poor neurological outcome is predominantly associated with diffuse brain damage and secondary hypoxic insults. Post-traumatic hypoxia is known to exacerbate primary brain injury; however, the underlying pathological mechanisms require further elucidation. Using a rat model of diffuse traumatic axonal injury (TAI) followed by a post-traumatic hypoxic insult, we characterized axonal pathology, macrophage/microglia accumulation, and astrocyte responses over 14 days. Rats underwent TAI alone, TAI followed by 30 min of hypoxia (TAI + Hx), hypoxia alone, or sham-operation (n = 6/group). Systemic hypoxia was induced by ventilating rats with 12% oxygen in nitrogen, resulting in a ∼ 50% reduction in arterial blood oxygen saturation. Brains were assessed for axonal damage, macrophage/microglia accumulation, and astrocyte activation at 1, 7, and 14 days post-treatment. Immunohistochemistry with axonal damage markers (ß-amyloid precursor protein [ß-APP] and neurofilament) showed strong positive staining in TAI + Hx rats, which was most prominent in the corpus callosum (retraction bulbs 69.8 ± 18.67; swollen axons 14.2 ± 5.25), and brainstem (retraction bulbs 294 ± 118.3; swollen axons 50.3 ± 20.45) at 1 day post-injury. Extensive microglia/macrophage accumulation detected with the CD68 antibody was maximal at 14 days post-injury in the corpus callosum (macrophages 157.5 ± 55.48; microglia 72.71 ± 20.75), and coincided with regions of axonal damage. Astrocytosis assessed with glial fibrillary acidic protein (GFAP) antibody was also abundant in the corpus callosum and maximal at 14 days, with a trend toward an increase in TAI + Hx animals (18.99 ± 2.45 versus 13.56 ± 0.81; p = 0.0617). This study demonstrates for the first time that a hypoxic insult following TAI perpetuates axonal pathology and cellular inflammation, which may account for the poor neurological outcomes seen in TBI patients who experience post-traumatic hypoxia.