27. Hypercapnea does not consistently increase the oxidation of cytochrome c oxidase in traumatic brain injury
I. Tachtsidis1, C. Pritchard2, M.M. Tisdall2, T.S. Leung1, C.E. Elwell1 and M. Smith2
1Medical Physics and Bioengineering, University College London; 2Department of Neuroanaesthesia and Neurocritical Care, The National Hospital for Neurology and Neurosurgery, London, UK
Background and aim: Using broadband near-infrared spectroscopy (BBS), we have previously demonstrated an increase in aerobic metabolism in the human healthy brain following hypercapnea and hyperoxia.1 We also observed oxidation in cerebral cellular and mitochondrial redox states, using cerebral microdialysis (MD) and BBS respectively, following hyperoxia in patients with traumatic brain injury (TBI).2 In this study we investigate the effect of hypercapnea on regional metabolism in the injured brain using BBS and MD.
Methods: Following ethics approval and assent from nominated representatives, 6 sedated patients with severe TBI were studied 3–5 days after injury. Cerebral blood flow velocity (Vmca) was measured using transcranial Doppler ultrasonography. Changes in cellular and mitochondrial redox state were measured from the changes in brain tissue lactate and pyruvate concentration using MD and the change in oxidized cytochrome c oxidase concentration ([oxCCO]) using BBS. [oxCCO] was measured in a region of interest adjacent to the site of the MD catheter. Intracranial pressure (ICP) was also monitored continuously. During a period of cardiovascular stability, the rate of mechanical ventilation was reduced in a stepwise manner to produce an increase in PaCO2 of approximately 1.5kPa. Changes in the continuously measured variables ([oxCCO], ICP and Vmca) during hypercapnea were compared with baseline values using a Student's paired t-test (significance at P<0.05).
Results: In all patients hypercapnea caused a significant increase in Vmca and ICP. Analysis of the BBS data demonstrated two patterns of [oxCCO] change in response to hypercapnea, Group A showing a significant increase in [oxCCO] and Group B a significant decrease (P<0.05).
Conclusions: In our previous study in TBI, hyperoxia resulted in an increase in [oxCCO] in all patients, consistent with an increase in aerobic metabolism secondary to the induced increase in cerebral oxygen delivery.2 Hypercapnea can also increase cerebral oxygen delivery via an increase in cerebral blood flow. In this study Vmca increased in all patients during hypercapnea. However despite the likely increase in cerebral oxygen delivery, we did not see an increase in the oxidation status of [oxCCO] in every patient. Interestingly, changes in MD measured metabolic variables were similar in all patients. Further work is required to determine why the continuous optically measured metabolic variable was able to identify two distinct patterns of change in [oxCCO] in response to hypercapnea whereas the non continuous MD variables were not.
Summary of results (LPR = lactate; pyruvate ratio)
P<0.05 for within group changes from baseline for continuously measured variables.
116. Anaesthetics influence closed head injury induced blood-brain barrier disruption, cerebral blood flow, brain edema and brain pathology
H.S. Sharma1, L. Wiklund1 and R. Patnaik1,2
1Surgical Sciences, Uppsala University Hospital, Uppsala, Sweden; 2Biomaterials, School of Bioengineering, Banaras Hindu University, Varanasi, India
Closed head injury (CHI) is a leading cause of death and induces severe neurological disability in surviving victims. In the United States alone the incidence of CHI admitted to hospitals is estimated to be at least 2,000 per million populations and more than 400,000 new cases are added each year in which large number of victims demonstrates significant long-term disabilities. The effect of anesthetics on neurological outcomes in head injury patients and the potential benefits of total systemic anesthesia compared with volatile gas anesthesia on various brain functions are still not been well evaluated. This investigation was undertaken to study the development of brain pathology and functional outcome following a well-established model of closed head injury (CHI) in rats under different intravenous or volatile gas anesthesia. Since previous works from our laboratory shows a profound rise of plasma and brain serotonin level following CHI, the effect of different anesthetics on plasma and brain serotonin level in relation to changes in blood-brain barrier (BBB) permeability, brained mea development, alteration in cerebral blood flow (CBF) and brain pathology was also evaluated.
The CHI was produced by an impact of 0.224 N on the right parietal bone under volatile ether anesthesia or systemic ketamine, pentobarbital or equithesin anaesthesia administered intraperitoneally. The CHI was inflicted by dropping a weight of 114.6 g on the skull from a height of 20 cm through a guide tube. This concussive brain injury resulted in profound leakage of Evans blue and radioiodine tracers in both the hemispheres and underlying sub cortical tissues that closely correspond to brain edema formation and volume swelling at 5 h after the CHI. These changes were most pronounced in the contralateral cerebral hemisphere. At this time a marked decrease in the regional CBF was seen in both the hemispheres that was most marked in the contralateral side. The plasma and brain serotonin showed a pronounced increase and exhibited a good correlation with the edema formation. Profound cell damage is seen in many parts of the brain at 5 h that are most marked in left uninjured hemisphere compared to the right injured side. These pathophysiological changes were most marked when the CHI was produced under ether anesthesia compared to systemic anesthesia. Mild but significantly less pathological changes are seen when the injury was made under ketamine aesthesia compared to pentobarbital anesthesia. The equithesin anesthesia showed moderate brain pathology quite comparable to that seen under pentobarbital anesthesia. Interestingly, the plasma and brain serotonin levels were highly correlated with the development of brain edema in animals subjected to CHI under various anesthetics. This suggest that anesthetic stress plays important roles in inducing serotonin levels in the brain and plasma following brain injury that could be detrimental in brain pathology. The functional outcome as seen using rota rod performances or grid walking following CHI were most adversely affected under ether anesthesia followed by pentobarbital, equithesin and ketamine. This indicates that that anesthetics are able to markedly influence the functional and pathological outcome of CHI.
292. LPS preconditioning attenuates neurobehavioral deficits following controlled cortical impact brain injury in mice
L. Longhi1, C. Perego2, N. Sacchi1, E. Zanier2, F. Ortolano1, N. Stocchetti1, T. Mcintosh3 and M.G. De Simoni2
1University of Milano; 2Mario Negri Institute, Milano, Italy; 3Media NeuroConsultants, Media, Pennsylvania, USA
Background and aims: To date it is unknown whether traumatic brain injury could benefit from preconditioning stimuli. We tested the hypothesis that a low dose of lipopolysaccharide (LPS) that is known to act as preconditioning stimulus in models of hypoxia/ischemia1 could attenuate the neurobehavioral sequelae of controlled cortical impact (CCI) brain injury.
Methods: C57/Bl6 mice (n = 12) were subjected to either intraperitoneal injection of LPS (0.1 mg/Kg)1 or saline. Subsequently they were anesthetized with sodium pentobarbital (65 mg/kg) and subjected to CCI brain injury2 at the following time points: 1, 3, 5, and 7 day-interval. Another group of mice received identical LPS injection, anesthesia and surgery, to serve as uninjured (sham-operated) controls. Neurobehavioral motor outcome was evaluated by a blinded investigator at 1 week postinjury by performing the Neuroscore.3
Purpose: To evaluate the effects of LPS on functional outcome following CCI brain injury.
Results: All brain-injured mice showed a robust behavioral deficit at 1 week postinjury compared to sham-operated mice. Mice receiving LPS at either 3, 5 and 7 day-interval prior to CCI brain injury had a behavioral performance that was significantly better compared to that of mice receiving saline or LPS at 1 day-interval (P<0.01 Kruskall-Wallis analysis of variance (ANOVA) followed by Mann-Whitney U test for individual comparison).
Conclusions: Our data show for the first time that the neurobehavioral sequelae of traumatic brain injury can be attenuated by a preconditioning stimulus.
504. Traumatic injury induces cell-specific shifts of branched chain amino acid metabolism in rat brain
M. Ren1, G. Xing2, W. Watson3, A. Verma3 and J.T. O'Neill1
1Pediatrics, Uniformed Services University of the Health Sciences Services; 2Psychiatry; 3Neurology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
Background: Traumatic brain injury (TBI) is associated with a primary immediate insult and secondary cascades of biochemical processes that can result in delayed cell death. Secondary injury of TBI presents an opportune therapeutic target for neuroprotection, but very rarely have potential research agents and techniques proven clinically beneficial making the search for novel agents all the more urgent. We suspect metabolism of branched chain amino acids (BCAAs) leucine, isoleucine, and valine could modify these injurious cascades. A further understanding of these pathways could lead to potential manipulation as a potent therapeutic strategy. Normally BCAAs are not only substrates for protein synthesis but are also essential for transamination reactions that yield an ammonium group which can buffer CNS glutamate and GABA levels. Transamination of BCAAs produces their branched chain keto-acid (BCKA) counterparts, which can be metabolized as bioenergetic fuels via the mitochondrial BCKA dehydrogenase (BCKD) complex. The BCKD is tightly regulated by BCKD kinase (BCKDK) and is inactivated upon phosphorylation. In response to pathological conditions the metabolism of BCAAs may adapt multiple roles for cell and tissue preservation.
Objectives: To assess modification of BCAA metabolic pathways by analyzing their associated enzymes (transaminase, dehydrogenase and kinase) and thereby determine the likely pathway BCAAs follow in response to traumatic injury. We also examined the activation state of BCKD in response to stress of primary cultured rat neurons and astrocytes.
Methods: Our TBI model was controlled cortical impact (CCI) in rats. CCI was induced with a penetration width of 6 mm, depth of 2.5 mm, and velocity of 4 m/s. Brains were analyzed at 4 hr, 1 d, 3 d and 7 d post TBI with immunohistochemistry, RT-PCR and western blot. Neurons and astrocytes were studied as primary cultures and analyzed for BCKD and pBCKD before and after metabolic stress.
Results: We found BCAA transaminases as well as BCKDK decreased 29% and 10%, respectively (P<0.05), 4 h following CCI. BCKDK then increased by 200% (P<0.05) by 3 days. The overall level of pBCKD increased 1 day (∼100%, P<0.05) after CCI. Immunohistochemical analysis demonstrated that pBCKD localized within neurons under normal conditions. However, following CCI, pBCKD dramatically increased in astrocytes, yet decreased in neurons. In rat brain primary culture, we found 2 fold (P<0.05) higher basal levels of BCKD in neurons than in astrocytes while the pBCKD levels were similar. When the astrocyte cultures were challenged with TGF-β there was a marked increase (125%, P<0.05) in pBCKD levels, while neuronal cultures treated with glutamate demonstrated decreased (69% P<0.05) in pBCKD levels.
Conclusions: Our findings demonstrate significant differences in the activation state of BCKD in neurons and astrocytes under normal and pathological conditions. Our results suggest reactive astrocytes may shift BCKAs away from mitochondrial utilization resulting in greater concentrations to supply neurons with more energy substrate after brain insults. Funded by NIH grant NS37814, and DoD grants MDA-905-02-2-0005 and MDA905-03-2-0001 (A.V. PI).
507. Low dose AT1 inhibition improves neurological outcome and reduces secondary brain damage 24 h after brain trauma in mice
R. Timaru-Kast, C. Ricken, K. Engelhard, C. Werner and S.C. Thal
Department of Anesthesiology, Johannes Gutenberg-University, Mainz, Germany
Objectives: The intrinsic renin-angiotensin system is capable to modulate brain injury. Angiotensin II receptor type 1 (AT1) mediates vasoconstriction, apoptosis and neuroinflammation, increasing secondary brain damage. Whereas angiotensin II receptor type 2 (AT2) triggers vasodilatation, anti-inflammation and neuro-proliferation that may offer protection.1 A recent study demonstrated a down-regulation of AT1 after controlled cortical impact (CCI) while AT2 was up-regulated temporarily.2 The present study explores the potential neuroprotective effect of AT1 inhibition on secondary brain damage after CCI. The first goal of the study is dosage finding of the specific AT1 antagonist candesartan. Secondly the time window for the effective dose of candesartan was investigated.
Methods: C57Bl6 mice were anesthetized with fentanyl, midazolam and medetomidine, subjected to a pneumatic brain trauma on the right parietal cortex (CCI) and randomly assigned to different treatment groups: Study I: vehicle solution (Na2CO3), low (0.1 mg/kg), medium (0.5 mg/kg) or high dose (1 mg/kg) candesartan (n = 8 each group, s.c. injection 30 min post CCI). Study II: application time points for candesartan 30 min, 1 h, 2 h or 4 h after CCI (n = 7 at each time point), or vehicle (n = 3 at each time point). Contusion volume was measured in Nissl stained sections 24 h after CCI. In both studies, the primary lesion (PrimL) was quantified in a separate set of animals 15 min after CCI (n = 8 and 7, respectively). Neurological outcome was assessed 24 h after CCI in Study II by Neurological Severity Score (NSS).
Results: Study I: 24 h after CCI contusion volume increased from 24 mm3 (PrimL) to 41 mm3 (vehicle). Low dose candesartan significantly reduced injury compared to vehicle (25 mm3, P<0.05), while medium and high dose candesartan did not significantly change lesion volume (35 and 36 mm3, respectively).
Study II: NSS revealed a reduction of neurofunctional deficits in animals treated with low dose candesartan at all application time points. Contusion volume increased from 22 mm3 (PrimL) to 47 mm3 (vehicle). At all application time points contusion volume was markedly reduced by app. 40%, when compared to vehicle (33 mm3, 36 mm3, 34 mm3 and 37 mm3 for 30 min, 1 h, 2 h and 4 h, respectively; P<0.05).
Conclusions: The present study demonstrates that low dose candesartan reduces neurofunctional impairment and secondary brain damage when applied for up to 4 h after experimental brain trauma. Possible mechanisms are:
direct vascular effects of AT1 receptor inhibition, i.e. increased cerebral blood flow by arterial vasodilatation, and
a shift of intracerebral angiotensin II action from AT1 mediated pro-apoptotic and inflammatory to AT2 mediated neuroproliferative and protective effects.
In accordance with previous ischemic stroke studies,3 higher doses candesartan failed to reduce lesion volume. Beneficial effects of high dose AT1 inhibition may be abolished by systemic side effects, e.g. reduction of mean arterial blood pressure. However, low dose AT1 inhibition is a promising therapeutic strategy to prevent secondary brain damage.
568. Controlled cortical impact induces expression of acid-sensing ion channel 2a in mouse brain
C. Goodman1, M. Van2, H. Saucedo Crespo2 and C. Robertson2
1Pathology; 2Neurosurgery, Baylor College of Medicine, Houston, Texas, USA
Background and aims: Acid-sensing ion channel (ASIC's) are ligand gated cation channels that open and flux sodium and calcium in response to increased proton concentration. In the periphery, these channels play a role in the detection of tissue ischemic injury, but they are also expressed in the central nervous system where their physiological role is unclear. ASIC's are up-regulated in ischemia and they may potentiate tissue damage in ischemia induced acidosis—a process that has been designated ‘acidotoxicity.’ ASIC knock-out animals are resistant to cerebral ischemia and there are numerous molecules capable of blocking these channels with beneficial effect. Since acidosis is a common neurochemical finding in traumatic brain injury, we examined the ASIC expression in experimental focal traumatic brain injury.
Methods: Under general anesthesia, C57BL6/J mice were exposed to moderate controlled cortical impact (CCI). ASIC 2a gene expression was ascertained by reverse transcription polymerase chain reaction (RT-PCR) and protein levels were measured by Western blotting at 2, 24, 48 and 72 h in the injured and uninjured cerebral hemispheres.
Results: No change in ASIC protein levels was seen at 2 h but by 24 h there was increased protein concentrations on the injured side. By 48 and 72 h, the ASIC protein levels were equal on injured and uninjured sides. Gene expression was increased at 2 h after CCI but by 24 h expression was equal on both sides.
Conclusions: ASIC 2a expression is rapidly increased in cerebral cortical tissue injured by controlled cortical impact. The rapidity of the increased expression is similar to that seen in ischemia but the expression is more transient in CCI. Acidosis is a common neurochemical feature of ischemic and traumatic brain injury; therefore, increased expression of these channels may play a role in the evolution of traumatic brain injury and the increased vulnerability of injured cerebral tissue to secondary injury. Studies in ischemic injury suggest that the ASIC's are potential therapeutic targets and the present study indicate that pharmacological modulation of these channels might also be useful in traumatic brain injury. Amiloride class drugs as well as several invertebrate neurotoxins are potent ASIC blockers that may have therapeutic potential in managing acidotoxicity in ischemia and traumatic brain injury.
Research funding support from the National Institutes of Health P01 Grant #NS38660 is gratefully acknowledged. All experiments were performed under appropriate approved institutional animal use protocols.
578. Neuroprotection by human umbilical mesenchymal cells transplatation after experimental traumatic brain injury
K.-F. Huang1,2, C.-W. Hsu3, Y.-S. Fu4 and J.-Y. Wang3
1Neurosurgery, Tzu Chi General Hospital; 2Medical Sciences; 3Physiology, National Defense Medical Center; 4Anatomy, National Yang-Ming University, Taipei, Taiwan, R.O.C.
Neuronal transplantation has provided a promising approach for treating traumatic brain injury (TBI). Human umbilical mesenchymal cells (HUMCs) have been suggested to promote survival of degenerating neurons after parkinsonism.
The purpose of this study was to evaluate the potential for survival, migration, differentiation and functional improvement of HUMCs transplanted into a rat model of TBI. Cortical impact injury (CCI) was produced in pentobarbitol-anaesthetised rats using a pneumatic piston with a 2.5-mm metal tip through a 5 mm parietal bone craniotomy and at a velocity of 4m/sec and a 2 mm deformation depth below the dura. HUMCs isolated from Wharton's jelly of the umbilical cord were injected into the ipsilateral or contralateral hippocampus of adult male rats 2 days following CCI. The animals were evaluated for neurological deficits using adhesive removal and rotorod tests at 1, 4, 7, 14 days and 1, 2, 3 months after transplantation. The animals were sacrificed at 1, 2 and 3 months after transplantation for immunohistochemistry evaluation. To study the proliferation and phenotypic differentiation of HMSCs, brain sections were immunostained for cell proliferation markers (bromodeoxyuridine. BrdU), or neuronal (NeuN), microglial (OX-42) and astrocytic (glial fibrillary acidic protein) markers. Behavioral testing revealed improvements in the rotorod test in ipsilateral HMSC-treated rats. The transplanted HMSCs successfully survived and migrated into injured brain and were preferentially localized around the injury site. Our results suggest that HMSCs have the potential for treatment of traumatically injured nervous system.
634. Neuroprotective and anti-inflammatory effects of histone deacetylase inhibition by valproate in traumatic brain injury
H. Rhinn1, V. Escriou1, D. Sherman1, E. Ake2, M. Plotkine2, C. Marchand-Leroux2 and V. Besson2
1U640; 2Pharmacology of Cerebral Circulation, EA2510, Paris Descartes University, Paris, France
Objectives: TBI leads to a deleterious neuroinflammation evidenced by oedema, cytokines production, matrix metalloproteinases (MMP) activation and leukocytes infiltration. As many inflammatory mediators are regulated at the transcriptional level, a pleiotropic action on their transcription may afford a massive anti-inflammatory effect. Histone deacetylases (HDAC) are a family of enzymes removing acetyl groups from histones and nuclear components such as transcription factors, which make them playing a central role in the transcription modulation. Recently, valproic acid (VPA), a well-known drug largely used for the treatment of seizures and bipolar mood disorders, has been reported to inhibit HDAC causing hyperacetylation.1 So we hypothesized that VPA could promote beneficial effects on deleterious consequences induced by TBI.
Methods: Male Swiss mice (28–32 g) anaesthetized under 2% halothane, were submitted to TBI using a weigh-drop device (50 g weight), as previsouly described.2 Mice were given VPA (300 mg/kg) or its vehicle (0.9% NaCl) intraperitoneally 5 min after TBI as this dose inhibits HDAC activity demonstrated by an increase of histone H3 acetylation.3 The neurological deficit was assessed by grip score measuring the length of time (in seconds) that mice remained on the string in some manner (using one or more paws, tail, tail plus paws), for a maximum of 30 seconds. In addition, during these 30-second period, the string test, scoring from 0 (severely impaired) to 5 (normal), evaluated the way mice could hang and move on the string. Cerebral oedema was determined by measuring brain water content (BWC) using the wet weight-dry weight technique. Levels of mRNA of 5 inflammatory mediators (interleukin-1β, chemokine receptor CCR2, MMP9 and 12, P-selectin) and the one of the chaperone protein HSP70 were quantified by quantitative RT-PCR. The effect of VPA on HDAC activity was evaluated by immunohistochemistry of acetylated histone H3.
Results: Uninjured mice had a grip score of 30±0.00 s and a string score of 5.0±0.00 at 24 h. At 24 h post-TBI, the grip and string scores were markedly decreased in vehicle-treated mice (grip: 19.4±3.03 s, P<0.01; string: 1.0±0.30, P<0.001) showing a neurological deficit. VPA-treated mice showed an improved grip (25.7±2.00 s, P<0.05) and string (2.5±0.52, P<0.05) scores after TBI, demonstrating a neurological recovery-promoting effect. The BWC of uninjured mice was 79.6%±0.11%. TBI induced an increase in the BWC (80.2%±0.18%, P<0.05) showing cerebral oedema. Treatment with VPA reduced the post-traumatic increase in BWC (79.7%±0.16%, P<0.05) demonstrating an anti-oedematous effect. The increase in levels of mRNA encoding for IL-1β, CCR2, MMP9 and 12, P selectin observed after TBI were reduced by VPA (P<0.001). Moreover, VPA increased levels of post-traumatic mRNA of HSP70 (P<0.01).
Conclusion: Our data show that VPA promotes neurological recovery and anti-oedematous effects after TBI. Beneficial effects induced by VPA are mediated, at least in part, through inhibition of HDAC, induction of HSP70 and reduction of levels of mRNA of 5 inflammatory mediators.
657. Combination therapy with Fenofibrate and simvastatin on deleterious consequences induced by traumatic brain injury
X.R. Chen, T. Beziaud, M. Plotkine, C. Marchand-Leroux and V. Besson
Pharmacology of Cerebral Circulation, EA2510, Paris Descartes University, Paris, France
Objectives: We1 and others2 have demonstrated that fibrates and statins, exerted neuroprotective and pleiotropic effects in experimental models of traumatic brain injury (TBI). As combination of statins and fibrates synergistically enhanced peoxisome proliferator-activated receptor (PPAR) α activation, we hypothesized the combination of both class of drugs exert more important and/or prolonged beneficial effects in TBI than each one alone. In this study, we examined the effect of the combination of fenofibrate and simvastatine on the consequences of TBI.
Methods: TBI was induced by lateral fluid percussion of the temporoparietal cortex on male Sprague-Dawley rats (300–330 g), as previously described.1 Sham-operated rats underwent the same surgery except for percussion. Simvastatin at 37.5 mg/kg, 50 mg/kg fenofibrate, and its combination or its vehicle (water containing 0.5% methylcellulose) were administrated by gavage 1 and 6 h after brain injury. At 24, 48 h, 3 and 7 days after TBI, a neurological assessment was done by 2 tests: one for reflexes and sensorimotor responses (ranging from 0 = worst to 9 = best), and the second untitled beam walking test for motor coordination (ranging from 0 = worst to 4 = best). Then rats were killed, and brain lesion was measured at 7 days post-injury.
Results: TBI led to a decrease in the neurological score at 24 h post-injury (P<0.001) demonstrating a neurological deficit that persisted at 48 h (P<0.001). Rats treated with fenofibrate (P<0.05), simvastatin (P<0.01) or their combination (P<0.05) showed an increase in the neurological scores at 24 h (fenofibrate: P<0.05; simvastatin: P<0.01; combination: P<0.01) showing a neurological recovery. Even if monotherapies had no more effects on the neurological score at 48 h post-TBI and later, combination still showed an improvement of the neurological score until, at least, 7 days after TBI (P<0.01) demonstrating a longest neurological recovery promoting effect. At 48 h and later after injury, rats receiving the combination showed an improvement of the beam walking score (P<0.05) whereas monotherapies had no effects. TBI induced a brain lesion of 83±8 mm3 that was reduced by treatment with the combination (36±12 mm3, P<0.05), whereas both monotherapies did not reduce it (fenofibrate: 62±20 mm3; simvastatin: 65±22 mm3).
Conclusions: Evidence that fenofibrate combined with simvastatin promote long-lasting beneficial effects on neurological recovery and lesion provides a strong basis for the use of this combination for TBI treatment.
742. Seizure susceptibility after traumatic brain injury is attenuated by hypothermia treatment
D. Dietrich1, C. Atkins1, J. Truettner1, G. Lotocki1, J. Sanchez-Molano1, O. Alonso1, T. Sick2 and H. Bramlett1
1Neurological Surgery; 2Neurology, University of Miami Miller School of Medicine, Miami, Florida, USA
Objectives: A significant, debilitating consequence of traumatic brain injury (TBI) is the development of seizures. TBI patients have a 3–4 fold higher risk of developing epilepsy than the general population.1 The pathomechanisms underlying posttraumatic seizures include hippocampal interneuron loss, hyperexcitability of dentate granule cells and abnormal sprouting of the mossy fiber pathway in the dentate gyrus2–5). Hypothermia treatment is a highly promising therapy that targets multiple pathomechanisms caused by TBI. The objective of this study was to determine if hypothermia reduces posttraumatic seizure susceptibility by attenuating the histopathological mechanisms contributing to seizure development.
Methods: Adult male Sprague Dawley rats received moderate parasagittal fluid-percussion brain injury (FPI) or sham surgery. The animals were maintained at normothermia (37.0°C–37.5°C) or hypothermia (33.0°C–34.0°C) for 4 hr starting 30 min after TBI. Moderate FPI does not elicit behaviorally evident seizures. Thus, to ascertain seizure susceptibility, animals received a subconvulsant dose of pentylenetetrazole (PTZ, 30 mg/kg), a GABAA receptor antagonist at 1 or 12 weeks post-injury. Seizure class and frequency were scored with simultaneous EEG recordings to assess for seizure threshold changes. Mossy fiber sprouting was determined by TIMM staining. In another set of animals, hyperexcitability changes of dentate granule cells were assessed in acute hippocampal slices by measuring input/output curves of the perforant pathway onto dentate granule cells.
Results: We found that moderate FPI resulted in increases in input/output curves of dentate granule cells at 1 week post-injury. This paralleled a decrease in seizure threshold in TBI animals as compared to naïve animals and this was reduced with hypothermia treatment. Similarly, at 12 weeks post-injury, seizure threshold was decreased and the highest seizure class reached was increased in TBI animals; these traumatic outcomes were prevented by hypothermia. These behavioral findings correlated with epileptic seizure discharges recorded by EEG. Post-hoc analysis revealed that mossy fiber sprouting of the dentate gyrus after TBI was reduced with hypothermia therapy.
Conclusions: These results show for the first time that hypothermia therapy after TBI reduces posttraumatic seizure susceptibility and abnormal mossy fiber sprouting in the dentate gyrus. These preclinical studies demonstrate the promising potential of therapeutic hypothermia in improving outcome in TBI patients and open a new therapeutic avenue for the treatment of posttraumatic seizures.
744. Acute and chronic metabolic alterations in the brain after cervical spinal cord injury
H. Bramlett1, L. Daniels1, G. Lotocki1, J. Truettner1, W. Zhao2, A. Marcillo1, O. Alonso1 and D. Dietrich1
1Neurological Surgery, University of Miami Miller School of Medicine; 2Biomedical Engineering, University of Miami, Miami, Florida, USA
Objectives: Spinal cord injury (SCI) is a devastating clinical condition resulting in complex neurological consequences including sensory, motor and autonomic dysfunction. Recently, supraspinal changes have been documented after SCI including circuit plasticity as well as neuronal atrophy and cell death.1 Previous studies from our laboratory have utilized 2-deoxyglucose autoradiographic techniques combined with somatosensory circuit activation for measuring local cerebral metabolic rates of glucose utilization (lCMRGLU) to understand the metabolic changes occurring in brain regions after injury.2 The purpose of this study was to assess baseline alterations in lCMRGLU occurring in spinal and supraspinal areas after acute and chronic cervical SCI and the metabolic responsiveness of the brain to vibrissae barrel-field circuit activation.
Methods: Adult male Wistar rats received moderate cervical SCI at C5 or sham laminectomy surgery. Forty-eight hours (n = 4/group) or 6 weeks (n = 6/group) after SCI, rats underwent 2-DG and vibrissae barrel-field circuit activation as previously described.2 Semi-serial coronal brain sections were cut while cervical spinal cord specimens were cut longitudinally. Autoradiographic images were analyzed as previously described2 with region of interest analysis conducted on multiple brain regions including the three relay stations (ipsilateral trigeminal medullary nuclear complex (TMC), contralateral ventrobasal thalamic nucleus (VPM), contralateral barrel field cortex (S1 & S2)) of the vibrissae barrel-field circuit.
Results: In the non-stimulated condition, acute SCI animals showed widespread patterns of decreased lCMRGLU as compared to sham. Patterns of decreased lCMRGLU ranged from 10%–40% reductions throughout multiple structures. In the 48 hr SCI animals, whisker stimulation resulted in normal increases in lCMRGLU within the barrel-field circuit relay stations as compared to uninjured animals. Interestingly, whisker stimulation also led to widespread increases in glucose metabolism outside of this circuit ranging from 20%–100% compared to sham. At 6wks after SCI, normal levels of stimulation induced increases in lCMRGLU were seen in the relay stations compared to sham. However, in these animals, an expansion of the S1/S2 cortical field as well as aberrant metabolic activation of brain regions including cingulate cortex and striatum was observed. Finally, in addition to supraspinal structures, local alterations were seen within the injured spinal cord at both time points. At the site of injury, moderate hypometabolism was present while rostral and caudal spinal cord segments showed evidence for hypermetabolism compared to sham.
Conclusions: These data show that the brain and SC undergoes a complex pattern of metabolic changes following moderate cervical SCI. Such metabolic changes could indicate alterations in circuit activation after acute and chronic spinal cord injury. The findings suggest that it may be important to consider these consequences of SCI when designing strategies including rehabilitation and axonal regeneration to enhance recovery after SCI.
827. Role of vasopressin V1 receptors for post-traumatic brain edema formation, cerebral blood flow, and secondary brain damage
R. Trabold, S. Krieg and P. Nikolaus
Department of Neurosurgery, University of Munich Medical Center—Grosshadern, Ludwig Maximilians University, Munich, Germany
Objective: Reductions of cerebral blood flow (CBF) and the formation of brain edema are still among of the most deleterious sequels of traumatic brain injury (TBI) and their pathophysiology is still not well understood. The aim of the current study was to investigate the effect of arginine-vasopressin (AVP, antidiuretic hormone, ADH) receptors, important regulators of water homeostasis and blood flow, on post-traumatic brain edema, cerebral blood flow, neurological function, and secondary brain damage.
Methods: Male C57/Bl6 mice (n = 8–10 per group) were randomized to six experimental groups and subjected to controlled cortical impact (CCI; 8 m/s, 1 mm). 5 min after trauma animals received the AVP V1-receptor antagonist SR-49059/g either systemically (10 μg/g) or intracerebroventricularly (icv, 40 ng/g). CBF was assessed immediately after trauma by laser Doppler fluxmetry while cerebral water content (wet-dry method), ICP, neurological function (beam walk), and contusion volume (histomorphometry) were assessed 24 h after the impact.
Results: The systemic inhibition of AVP V1 reduced post-traumatic (30 min) by 26% as compared to controls (P<0.05) while the central application had no effect on CBF. Further experiments using the icv protocol reduced post-traumatic brain edema formation by 68% (P<0.05), intracranial hypertension by 46% (P<0.05), secondary contusion expansion by 46% (P<0.05), and significantly improved the neurological function (P<0.05).
Conclusion: The current results demonstrate that vasopressin V1 receptors are involved in the pathogenesis of post-traumatic brain damage. Although the underlying mechanisms of AVP-mediated brain edema formation and CBF remain to be elucidated, our study suggests that vasopressin V1 receptors may represent a novel pharmacological target for the treatment of secondary brain damage following traumatic brain injury.
901. Localized temperature dynamics during pharyngeal selective brain cooling
D. Coman1,2, H.K.F. Trubel3,4, P. Herman1,2,5 and F. Hyder1,2,6
1Diagnostic Radiology; 2Quantitative Neuroscience with Magnetic Resonance (QNMR), Yale University, New Haven, Connecticut, USA; 3Department of Pediatrics, University Witten/Herdecke, HELIOS-Klinikum Wuppertal; 4Bayer HealthCare Research Center, Wuppertal, Germany; 5Semmelweis University, Budapest, Hungary; 6Biomedical Engineering, Yale University, New Haven, Connecticut, USA
Introduction: Recently, we described a new approach to obtain pharyngeal selective brain cooling (pSBC).1,2 In this method a cooling coil is inserted into the pharynx in order to cool the brain selectively. Temperature distributions in rat brain can be obtained within minutes by using a new temperature-sensitive probe which is based on the complex between thulium ion (Tm3+) and the macrocyclic chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetramethyl-1,4,7,10-tetraacetate or DOTMA4−.3 In the present study we use TmDOTMA− agent to measure time-dependent temperature distributions and we calculate the corresponding cooling rate constants in rat brain.
Materials and methods: Animal preparation: Sprague-Dawley rats (220–315 g) were tracheotomized and artificially ventilated (70% N2O, 30% O2). During the animal preparation, isoflurane (1% to 2%) was used for induction. The anesthetized rats were prepared with renal ligation as previously described.4 pSBC was achieved by running ice-cold water for 2 h through the cooling coil inserted into the pharynx. In vivo (n = 4): A gaussian pulse of 200 ms was used for excitation of a 6 mm slice with FOV of 2.56 cm × 2.56 cm. The following parameters were used: 16×16 encode steps, TR = 11 ms, 100 averages and 4 min 40 s acquisition time. The temperature maps were calculated from the chemical shifts of TmDOTMA− methyl group according to the equation: T = 346+4.6·δCH3+0.0152·δCH32. For each animal, the cooling and the recovering rate constants were calculated by fitting the temperature variation over time to a single exponential.
Results: The results for all the animals investigated indicate that there is a negative correlation (R = 0.88) between the average cooling rate constants (kc) and the average recovering rate constants (kr) in each animal. This correlation is probably mediated by the local net heat contribution, which includes metabolic heat production, heat due to changes in cerebral blood flow or conductive heat exchange with the neighboring regions.5 The temperature maps show a relatively homogeneous temperature distribution across the entire cortical region, with standard deviations of less than 0.4°C. The distribution of the cooling rate constants is also relatively homogeneous within the same animal, although somewhat larger variations are observed between different animals. In each animal, the recovering rate constants show a distribution which is slightly more dispersed than that of the cooling rate constants. However, the range of the recovering rate constants for different animals investigated is relatively small (1 to 1.5 h−1), compared with a relatively larger range for the cooling rate constants (0.3 to 1.43 h−1).
Conclusion: Our results indicate that the pSBC rate constants are tightly dependent on the local net heat contribution, measured indirectly by the recovering rate constants.
Acknowledgements: Supported in part by P30 NS052519 of the QNMR Program.
902. MDMA-induced temperature changes and dynamics in rat brain
D. Coman1,2, L. Jiang1, F. Hyder1,2,3 and K. Behar4
1Diagnostic Radiology; 2Quantitative Neuroscience with Magnetic Resonance (QNMR); 3Biomedical Engineering; 4Department of Psychiatry, Yale University, New Haven, Connecticut, USA
Objectives: 3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a heavily abused psychostimulant which has seen explosive growth in its use during the last decade.1 The most severe and potentially fatal acute effects of MDMA involve extreme hyperthermia and its consequences on multiple organ systems, e.g., rhabdomyolysis, coagulopathy, kidney, heart, and liver failure.2,3 The magnitude, distribution, and dynamics of MDMA induced temperature changes are thus of critical importance in neurotoxicity, where core body temperature is most frequently reported. Recently, we showed that temperature distributions in rat brain can be obtained within minutes by using a new temperature-sensitive probe which is based on the complex between the thulium ion (Tm3+) and the macrocyclic chelate 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetramethyl-1,4,7,10-tetraacetate or DOTMA4−.4 In the present study we use the TmDOTMA− agent to measure time-dependent temperature distributions and from these we calculate the maps of temperature changes and those of MDMA-induced warming rate constants in rat brain.
Methods: Animal preparation: Sprague-Dawley rats (250–300 g) were tracheotomized and artificially ventilated (30% O2). The animals were anesthetized with an intraperitonial injection of urethane (1.3 g/Kg). The anesthetized rats were prepared with renal ligation as previously described.4 TmDOTMA− was continuously infused for ∼2 h, followed by the MDMA injection. In vivo (n = 3): A gaussian pulse of 200 ms was used for excitation of a 6 mm slice with FOV of 2.56 cm × 2.56 cm. The following parameters were used: 16×16 encode steps, TR = 11 ms, 100 averages and 4 min 40 s acquisition time. The temperature maps were calculated from the chemical shifts of TmDOTMA− methyl group according to the equation: T = 346+4.6·δCH3+0.0152· δ2CH3. For each animal, the MDMA-induced warming rate constants were calculated by fitting the temperature variation over time to a single exponential function.
Results: The results indicate a relatively homogenous distribution of warming rate constants, with subcortical regions showing slightly faster temperature increase than the cortical regions. The average rate constant of MDMA-induced temperature change was 1.05±0.13 h−1, while the average temperature change was 2.1°C±0.3°C. Although the distributions of both warming rate constants and temperature changes are relatively homogenous within the same animal, somewhat larger variations are observed when comparing different animals. The average warming rate constant was 1.4±0.4 h−1, while the average temperature change was +1.7°C±0.4°C. On average, the brain temperature increased from 35.5°C±0.2°C to 37.2°C±0.5°C, while the core temperature increased from 37.4°C±0.4°C to 39.8°C±0.4°C.
Conclusion: The current results indicate that the temperature increase in the rat brain after a dose of 20 mg/kg MDMA is relatively homogenous, with subcortical regions showing slightly faster temperature increase than the cortical regions.
Acknowledgenments: Supported in part by a pilot grant from P30 NS052519 of the QNMR Program.
960. Analysis of regional hypoperfusion in traumatic brain injury using 123I-IMP SPECT with 3D-SSP
T. Hayakawa, Y. Takasato, H. Masaoka, N. Otani, Y. Yoshino, H. Yatsushige, T. Sugawara, T. Momose, C. Aoyagi and G. Suzuki
Neurosurgery, National Disaster Medical Center, Tokyo, Japan
Objective: Cognitive function after head injury is important for the patients to be reintegrated in the society, but the lesion on conventional CT/MRI does not exactly correlate the neuropshychological impairment. In the previous studies, experience with SPECT in the assessment of head injury has shown that SPECT may reveal abnormalities that are not detected by CT or MRI. Three-dimensional stereotactic surface projection (3D-SSP) is one of the methods of statistical analysis, that allows more objective and clearer detection of decreased regional CBF than conventional SPECT analysis. This study investigated regional cerebral blood flow (CBF) in head injured patients using 3D-SSP to detect hypoperfusion on 123I-IMP SPECT scans.
Methods: 39 patients with traumatic brain injury (TBI) who underwent 123I-IMP SPECT at discharge (mean Day 36.7) were included in this study. Patients were divided into three groups according to the main lesion on CT scans; DAI group(diffuse axonal injury): 12cases, mean age 28.0, mean GCS score on admission 8.8, ASEDH group(acute subdural or epidural hematoma): 13 cases, mean age 37.1, mean GCS 11.2, Contusion group(cerebral contusion or contusional intracerebral hematoma): 14 cases, mean age 44.4, mean GCS 10.1. The SPECT images were analyzed statistically using 3D-SSP with Stereotactic extraction estimation (SEE) program.
Results: DAI group displayed low blood flow extensively in the basal frontal lobes (rectal gyrus, orbital gyrus) and the medial frontal lobes (i.e. anterior cingulate). This group also showed CBF decrease in the basal temporal lobes (parahippocampal gyrus, uncus) and the lateral frontal lobes (middle and inferior frontal gyrus). Examination of ASEDH group indicated the involvement of the contralateral frontal lobe in addition to the ipsilateral temporal and parietal lobe. In Contusion group, many patients showed CBF decrease in the basal frontal lobes and the basal temporal lobes in addition to the contusional region on CT. The hypoperfusion was common in the basal frontal lobes, medial frontal lobes and basal temporal lobes. In any type of the lesion on CT.
Conclusion: The SPECT analysis with 3D-SSP demonstrated hypoperfusion in many area where CT/MRI showed no abnormalities in TBI patients. The CBF reduction in the basal frontal lobes, medial frontal lobes and basal temporal lobes was found in any type of head injury, so this may be the characteristic pattern in TBI. 3D-SSP is useful in detecting functional abnormalities in TBI.
References
1.
TachtsidisI. Advances in Experimental Medicine and Biology2009;645:315–20.
2.
TisdallMM. Journal of Neurosurgery2008;109:424–32.
XiongZG. Acid-sensing ion channels (ASICs) as pharmacological targets for neurodegenerative diseases. Curr Opin Pharmacol2008;8(1):25–32.
10.
SimonR and XiongZ. Acidotoxicity in brain ischaemia. Biochem Soc Trans, 2006;34(Pt 6):1356–61.
11.
JohnsonMB. Global ischemia induces expression of acid-sensing ion channel 2a in rat brain. J Cereb Blood Flow Metab2001;21(6):734–40.
12.
Phiel. J Biol Chem2001;276:36734–41.
13.
Hellal. J Neurotrauma2003;20:841–51.
14.
Ren. J Neurochem2004;89:1358–67.
15.
Chen. J Neurotrauma2007;24(7):1119–31.
16.
Lu. J Neurotrauma2007;24(7):1132–46.
17.
GargaNLowensteinDH. Posttraumatic epilepsy: A major problem in desperate need of major advances. Epilepsy Curr2006;6:1–5.
18.
D'AmbrosioRFairbanksJPFenderJSBornDEDoyleDLMillerJW. Post-traumatic epilepsy following fluid percussion injury in the rat. Brain2004;127:304–14.
19.
GolaraiGGreenwoodACFeeneyDMConnorJA. Physiological and structural evidence for hippocampal involvement in persistent seizure susceptibility after traumatic brain injury. J Neurosci2001;21:8523–37.
20.
GradyMSCharlestonJSMarisDWitgenBMLifshitzJ. Neuronal and glial cell number in the hippocampus after experimental traumatic brain injury: Analysis by stereological estimation. J Neurotrauma2003;20:929–41.
21.
SanthakumarVRatzliffADJengJTothZSolteszI. Long-term hyperexcitability in the hippocampus after experimental head trauma. Ann Neurol2001;50:708–17.
22.
BramlettHMDietrichWD. Progressive damage after brain and spinal cord injury: Pathomechanisms and treatment strategies. Prog Brain Res2007;161:125–41.
23.
PassineauMJZhaoWBustoRDietrichWDAlonsoOLoorJYBramlettHMGinsbergMD. Chronic metabolic sequelae of traumatic brain injury: Prolonged suppression of somatosensory activation. Am J Physiol Heart Circ Physiol2000;279:H924–31.
