Jessie S. Truettner

Interleukin-1β messenger ribonucleic acid and protein levels after fluid-percussion brain injury in rats: Importance of injury severity and brain temperature

OBJECTIVE Posttraumatic temperature manipulations have been reported to significantly influence the inflammatory response to traumatic brain injury (TBI). The purpose of this study was to determine the temporal and regional profiles of messenger ribonucleic acid (mRNA) expression and protein levels for the proinflammatory cytokine interleukin-1&bgr; (IL-1&bgr;), after moderate or severe TBI. The effects of posttraumatic hypothermia (33°C) or hyperthermia (39.5°C) on these consequences of TBI were then determined. METHODS Male Sprague-Dawley rats underwent fluid-percussion brain injury. In the first phase of the study, rats were killed 15 minutes or 1, 3, or 24 hours after moderate TBI (1.8–2.2 atmospheres), for reverse transcription-polymerase chain reaction analysis. Other groups of rats were killed 1, 3, 24, or 72 hours after moderate or severe TBI (2.4–2.7 atmospheres), for protein analysis. In the second phase, rats underwent moderate fluid-percussion brain injury, followed immediately by 3 hours of posttraumatic normothermia (37°C), hyperthermia (39.5°C), or hypothermia (33°C), and were then killed, for analyses of protein levels and mRNA expression. Brain samples, including cerebral cortex, hippocampus, thalamus, and cerebellum, were dissected and stored at −80°C until analyzed. RESULTS The findings indicated that mRNA levels were increased (P < 0.05) as early as 1 hour after TBI and remained elevated up to 3 hours after moderate TBI. Although both moderate and severe TBI induced increased levels of IL-1&bgr; (P < 0.05), increased protein levels were also noted in remote brain structures after severe TBI. Posttraumatic hypothermia attenuated IL-1&bgr; protein levels, compared with normothermia (P < 0.05), although the levels remained elevated in comparison with sham values. In contrast, hyperthermia had no significant effect on IL-1&bgr; levels, compared with normothermic values. Posttraumatic temperature manipulations had no significant effect on IL-1&bgr; mRNA levels. CONCLUSION Injury severity determines the degree of IL-1&bgr; protein level elevation after TBI. The effects of posttraumatic hypothermia on IL-1&bgr; protein levels (an important mediator of neurodegeneration after TBI) may partly explain the established effects of posttraumatic temperature manipulations on inflammatory processes after TBI.

Tumor Necrosis Factor α Expression and Protein Levels after Fluid Percussion Injury in Rats: The Effect of Injury Severity and Brain Temperature

OBJECTIVE:Tumor necrosis factor &agr; (TNF&agr;) is elevated in some models of traumatic brain injury (TBI). However, it is unclear how TNF&agr; messenger ribonucleic acid (mRNA) expression and protein levels are affected by injury severity and posttraumatic temperature modification. This study determined the regional and temporal profile of TNF&agr; levels after moderate and severe TBI and assessed the effects of posttraumatic hypothermia or hyperthermia on this proinflammatory cytokine. METHODS:Adult male Sprague-Dawley rats were subjected to sham procedures (no injury), moderate fluid-percussion TBI (1.8–2.2 atm), or severe fluid-percussion TBI (2.4–2.6 atm). After 1 to 72 hours of survival, animals were killed, and brain samples, cerebrospinal fluid, and serum were harvested for enzyme-linked immunosorbent assay quantification of TNF&agr; levels. In a subsequent study, a 3-hour period of post-traumatic hypothermia (33°C) or hyperthermia (39.5°C) was applied, followed by immediate killing and cytokine assay. Another group was subjected to moderate TBI (1.8–2.2 atm), followed by killing at 15 minutes or at 1, 3, or 24 hours for TNF&agr; reverse transcriptase-polymerase chain reaction analysis. RESULTS:A significant increase in TNF&agr; mRNA and protein levels in cellular lysates of injured cortex and ipsilateral hippocampus was noted by 1 hour after TBI; it was sustained to 3 hours, followed by a rapid decline. Increased injury severity was associated with increased protein levels at remote injury sites and in the injured cerebral cortex at 72 hours. Posttraumatic hypothermia significantly reduced TNF&agr; mRNA expression in the hippocampus compared with that in normothermic rats. In contrast, no temperature effects on TNF&agr; protein levels were documented. CONCLUSION:Rapid and marked increase in TNF&agr; mRNA expression and protein levels follows moderate and severe TBI. Injury severity and posttraumatic temperature play a modest but significant role on TNF&agr; expression and protein levels. These findings suggest that the effects of posttraumatic temperature on histopathological and behavioral outcome primarily may involve secondary mediators that do not operate directly through their effect on TNF&agr;.

Effect of ischemic preconditioning on the expression of putative neuroprotective genes in the rat brain.

Previous studies have demonstrated that sublethal ischemic insults protect from subsequent ischemia in the intact brain. There are two windows for the induction of tolerance by ischemic preconditioning (IPC). One occurs within 1 h following IPC, and the other one develops from 1 to 3 days after IPC. The goal of this study was to determine whether IPC neuroprotection may be mediated by expression of known neuroprotective genes and to characterize the temporal and spatial expression patterns of these genes. IPC was produced by bilateral carotid artery occlusions and hypotension (50 mmHg) for 2 min. After various survival times, the expression of MAP-2, brain-derived neurotrophic factor (BDNF), c-jun, c-fos, nerve growth factor (NGF) and HSP70 was assessed by in situ hybridization of coronal brain sections with 35S labeled probes. BDNF, NGF, and c-jun were significantly upregulated in the hippocampus. c-fos was detected in the hippocampus, cortex and striatum. HSP70 mRNA was induced in the cortex, hippocampus, striatum, and thalamus. MAP-2 showed no change in expression, confirming previous studies that no cell death occurs following IPC. The increase in expression of these stress-related, neurotrophic and immediate early genes in response to a mild preconditioning insult may help mediate the protection of vulnerable neurons to subsequent lethal ischemic insults.

Influence of therapeutic hypothermia on matrix metalloproteinase activity after traumatic brain injury in rats

Recent evidence suggests that matrix metalloproteinases (MMPs) contribute to acute edema and lesion formation following ischemic and traumatic brain injuries (TBI). Experimental and clinical studies have also reported the beneficial effects of posttraumatic hypothermia on histopathological and behavioral outcome. The purpose of this study was to determine whether therapeutic hypothermia would affect the activity of MMPs after TBI. Male Sprague-Dawley rats were traumatized by moderate parasagittal fluid-percussion (F-P) brain injury. Seven groups (n = 5/group) of animals were investigated: sham-operated, TBI with normothermia (37°C), and TBI with hypothermia (33°C). Normothermia animals were killed at 4, 24, 72 h and 5 days, and hypothermia animals at 24 or 72 h. Brain temperature was reduced to target temperature 30 mins after trauma and maintained for 4 h. Ipsilateral and contralateral cortical, hippocampal, and thalamic regions were analyzed by gelatin and in situ zymography. In traumatized normothermic animals, TBI significantly (P<0.005) increased MMP-9 levels in ipsilateral (right) cortical and hippocampal regions, compared with contralateral or sham animals, beginning at 4 h and persisting to 5 days. At 1, 3, and 5 days after TBI, significant increases in MMP-2 levels were observed. In contrast to these findings observed with normothermia, posttraumatic hypothermia significantly reduced MMP-9 levels. Hypothermic treatment, however, did not affect the delayed activation of MMP-2. Clarifying the mechanisms underlying the beneficial effects of posttraumatic hypothermia is an active area of research. Posttraumatic hypothermia may attenuate the deleterious consequences of brain trauma by reducing MMP activation acutely.

Therapeutic hypothermia alters microRNA responses to traumatic brain injury in rats

Therapeutic hypothermia promotes protection after traumatic brain injury (TBI). The mechanisms underlying hypothermic protection are multifactorial and may include the modulation of microRNA (miRNA) expression after trauma. We utilized microarrays to examine the effects of posttraumatic hypothermia on the expression of 388 rat miRNAs. Animals were subjected to sham or moderate fluid percussion brain injury, followed by 4 hours of hypothermia (33°C) or normothermia (37°C) and euthanized at 7 or 24 hours. At 7hours, 47 miRNAs were significantly different (P < 0.05) between TBI and sham (15 higher in TBI and 31 lower). After 24hours, 15 miRNAs differed by P < 0.05 (7 higher and 9 lower). The expression of miRNAs was altered by posttraumatic hypothermia. At 7hours, seven were higher in hypothermia than normothermia and five were lower. Some miRNAs (e.g., miR-874 and miR-451) showed the most difference with hypothermia, with changes verified by quantitative reverse transcriptase-PCR. Regionally specific miRNAs also showed responses to TBI and hypothermia treatments by in situ hybridization. In addition, in vitro neuronal stretch injury studies showed similar temperature-sensitive responses to specific miRNAs. These novel data indicate that the reported beneficial effects of early hypothermia on traumatic outcome may include temperature-sensitive miRNAs involved in basic cell-processing events.

Subcellular stress response and induction of molecular chaperones and folding proteins after transient global ischemia in rats

Brain ischemia induces the toxic accumulation of unfolded proteins in vulnerable neurons. This cellular event can trigger the unfolded protein response (UPR) and activate the expression of a number of genes involved in pro-survival pathways. One of the pro-survival pathways involves the sequestration and elimination of misfolded and aggregated proteins. Recent evidence suggests that the endoplasmic reticulum (ER), mitochondria, and cytoplasm respond individually to the accumulation of unfolded proteins by induction of organelle specific molecular chaperones and folding enzymes. This study utilized a rat model of transient (15 min) global ischemia (2-vessel occlusion) to investigate the regional and temporal induction of some of these key stress proteins after ischemia. Electron microscopy demonstrated that visible protein aggregates accumulated predominately in the cytoplasm. We used in situ hybridization (forebrain structures) and western blot (hippocampus) analysis to measure changes in expression of heat shock protein 70 (HSP70 cytoplasmic), HSP60 (mitochondrial), ER luminal proteins glucose response proteins GRP78 and GRP94, protein disulphide isomerase (PDI), homocysteine-inducible, endoplasmic reticulum stress-inducible protein (HERP), and calnexin. Induction of mRNA for HSP70 occurred earlier (beginning at 30 min) and at a higher level relative to the delayed (4-24 h) and more moderate induction of mRNAs for mitochondrial matrix HSP60 and the ER lumen HERP, GRP78, GRP94, calnexin and PDI. Increases in hippocampal proteins were observed at 4 h (HSP70) and 24 h (HSP60, GRP78, GRP94) after reperfusion. These results demonstrate that after a transient ischemic insult, the subcellular responses to the accumulation of unfolded proteins varies between cellular compartments and are most prevalent in the cytoplasm and, to a lesser degree, in the mitochondrial matrix and ER lumen.

Systemic inflammation exacerbates behavioral and histopathological consequences of isolated traumatic brain injury in rats.

The proinflammatory cytokine interleukin-1beta (IL-1beta) is induced rapidly after traumatic brain injury (TBI) and contributes to the inflammatory events that lead to neuronal loss. Although an important source of IL-1beta is from the injured brain itself, in patients with multiple organ trauma (polytrauma) IL-1beta is also released into the bloodstream which may potentially influence brain vulnerability. The purpose of this study was to determine the effects of systemic inflammation induced by peripheral administration of IL-1beta on histopathological and behavioral outcome after moderate fluid percussion (FP) brain injury in rats. At 30 min or 24 h after TBI, saline, 20 mug/kg or 40 mug/kg of IL-1beta was injected (n=4-9/group) intraperitoneally (IP). Sham operated animals (n=9) received either saline or IL-1beta (20 or 40 mug/kg) injections. The somatosensory tactile placing test was administered at 1, 2 and 3 days posttrauma. IL-1beta-treated animals showed significant placing deficits compared to vehicle-treated TBI animals. Three days after injection, contusion areas and volumes were significantly increased (p<0.05) with both IL-1beta doses and at both treatment times compared to vehicle-treated animals. IL-1beta-treated rats showed more contusion injury and hippocampal neuronal damage as well as enhanced perivascular neutrophil accumulation. Cortical IL-1r1 mRNA increased as early as 1 h following TBI, peaking at 24 h and remained elevated 3 days posttrauma. These data show that the posttraumatic administration of IL-1beta significantly aggravates behavioral outcome and increases overall contusion volume after TBI. Increased systemic inflammatory processes, including extravasation of activated leukocytes and proinflammatory cytokines could participate in this detrimental outcome. Because peripherally circulating cytokines and other neurotoxic factors may be increased following multi-organ trauma, these findings may be important in targeting therapeutic interventions in this patient population.

Post-traumatic seizure susceptibility is attenuated by hypothermia therapy

Traumatic brain injury (TBI) is a major risk factor for the subsequent development of epilepsy. Currently, chronic seizures after brain injury are often poorly controlled by available antiepileptic drugs. Hypothermia treatment, a modest reduction in brain temperature, reduces inflammation, activates pro‐survival signaling pathways, and improves cognitive outcome after TBI. Given the well‐known effect of therapeutic hypothermia to ameliorate pathological changes in the brain after TBI, we hypothesized that hypothermia therapy may attenuate the development of post‐traumatic epilepsy and some of the pathomechanisms that underlie seizure formation. To test this hypothesis, adult male Sprague Dawley rats received moderate parasagittal fluid‐percussion brain injury, and were then maintained at normothermic or moderate hypothermic temperatures for 4 h. At 12 weeks after recovery, seizure susceptibility was assessed by challenging the animals with pentylenetetrazole, a GABAA receptor antagonist. Pentylenetetrazole elicited a significant increase in seizure frequency in TBI normothermic animals as compared with sham surgery animals and this was significantly reduced in TBI hypothermic animals. Early hypothermia treatment did not rescue chronic dentate hilar neuronal loss nor did it improve loss of doublecortin‐labeled cells in the dentate gyrus post‐seizures. However, mossy fiber sprouting was significantly attenuated by hypothermia therapy. These findings demonstrate that reductions in seizure susceptibility after TBI are improved with post‐traumatic hypothermia and provide a new therapeutic avenue for the treatment of post‐traumatic epilepsy.

Transient changes of brain-derived neurotrophic factor (BDNF) mRNA expression in hippocampus during moderate ischemia induced by chronic bilateral common carotid artery occlusions in the rat

Chronic bilateral common carotid artery occlusion (BCCAO) induces moderate ischemia (oligemia) in the rat forebrain in the absence of overt neuronal damage. In situ hybridization for brain-derived neurotrophic factor (BDNF) mRNA was used to search for a molecular response to moderate ischemia. BDNF mRNA was significantly increased in the hippocampal granule cells at 6 h of occlusion (ANOVA, Tukey test P<0.05). At 1, 7 and 14 days BDNF mRNA levels returned to control levels. The frequency of BDNF gene expression at 6 h was 83%, which was significantly higher than the 7% incidence of histological injury in the hippocampus (Fishers exact test, P<0.002). Cerebral blood flow was reduced to 75% of control levels in the hippocampus after 1 week of BCCAO when measured with the autoradiographic method. Measurements of tissue flow with a microprobe for laser Doppler flow excluded decreases into the ischemic range during the period when elevated gene expression was observed. Prolonged moderate ischemia (oligemia) is a sufficient stimulus for BDNF gene expression in the hippocampus. These molecular studies provide direct evidence for an involvement of the hippocampus in the BCCAO model.

Age-Dependent Changes in the Proteome Following Complete Spinal Cord Transection in a Postnatal South American Opossum (Monodelphis domestica)

Recovery from severe spinal injury in adults is limited, compared to immature animals who demonstrate some capacity for repair. Using laboratory opossums (Monodelphis domestica), the aim was to compare proteomic responses to injury at two ages: one when there is axonal growth across the lesion and substantial behavioural recovery and one when no axonal growth occurs. Anaesthetized pups at postnatal day (P) 7 or P28 were subjected to complete transection of the spinal cord at thoracic level T10. Cords were collected 1 or 7 days after injury and from age-matched controls. Proteins were separated based on isoelectric point and subunit molecular weight; those whose expression levels changed following injury were identified by densitometry and analysed by mass spectrometry. Fifty-six unique proteins were identified as differentially regulated in response to spinal transection at both ages combined. More than 50% were cytoplasmic and 70% belonged to families of proteins with characteristic binding properties. Proteins were assigned to groups by biological function including regulation (40%), metabolism (26%), inflammation (19%) and structure (15%). More changes were detected at one than seven days after injury at both ages. Seven identified proteins: 14-3-3 epsilon, 14-3-3 gamma, cofilin, alpha enolase, heart fatty acid binding protein (FABP3), brain fatty acid binding protein (FABP7) and ubiquitin demonstrated age-related differential expression and were analysed by qRT-PCR. Changes in mRNA levels for FABP3 at P7+1day and ubiquitin at P28+1day were statistically significant. Immunocytochemical staining showed differences in ubiquitin localization in younger compared to older cords and an increase in oligodendrocyte and neuroglia immunostaining following injury at P28. Western blot analysis supported proteomic results for ubiquitin and 14-3-3 proteins. Data obtained at the two ages demonstrated changes in response to injury, compared to controls, that were different for different functional protein classes. Some may provide targets for novel drug or gene therapies.