Patrick M. Kochanek - US grants

Head injury is an important clinical problem which is poorly understood and often unsuccessfully treated. Rodent models of head injury (such as the controlled cortical contusion model) have been developed that produce many of the features of human head injury. Using a given model, the importance of standardization methodologies in the investigation of the pathobiology and treatment of experimental traumatic brain injury is well recognized. We propose to operate a CORE facility that addresses the following SPECIFIC AIMS: 1) To provide a centralized facility for surgery and anesthesia for all of the proposed experimental manipulations of rats within this program project grant (PPG), 2) To provide uniform traumatic injury for all of the rat studies in the PPG (controlled cortical contusion model), and to ensure consistency of the insult severity by performing monthly quality control analysis, 3) To provide a centralized facility for the standardized assessment of both traumatic lesion volume and percent brain water (%BW) for all experiments within the PPG, 4) To facility the interaction between each of the PIs in the PPG (University of Pittsburgh Head Injury Research Center) and the collaborating investigators at the Pittsburgh NMR Center for Biomedical Research (Carnegie Mellon University), and 5) To provide a standardized hypothermia treatment regimen for all studies utilizing this therapeutic intervention in rats in the PPG. This CORE facility will be used by the following principal investigators involved in the individual proposals of this PPG: 1) Alan Palmer, Ph.d., "Excitatoxicity and free radical formation in brain trauma,' 2) Steven DeKosky, M.D., "Cytokines and neurotropic factors in traumatic brain injury," and 3) Patrick Kochanek, M.D., "Neutrophils and the acute inflammatory response to traumatic brain injury." To achieve these Specific Aims, a CORE laboratory has been established and specific protocols have been developed for the anesthesia and surgery involved in producing trauma in rats by controlled cortical contusion. Detailed protocols have also been developed for evaluation of traumatic lesion volume, and %BW using T2-weighted and proton density magnetic resonance imaging, respectively. In addition, a standardization hypothermia treatment regimen has been developed. Many of these protocols were used for the generation of the preliminary reports in this PPG. The successful execution of these Specific Aims will provide for optimal resource utilization by the investigators, including use of 1) technician time, 2) laboratory space, 3) equipment, 4) supplies, 5) rats, and 6) MRI time. At the same time, uniformity of the choice of the model among the investigators and quality control will also be ensured for all rat studies within the PPG. Finally, this CORE will facilitate both the interpretation of the findings of each PI and the interaction between PIs within the PPG. It will similarly facilitate comparison of our results with those from other laboratories nationally.

Secondary processes exacerbate brain injury after trauma. Recent studies suggest that neutrophils in the local acute inflammatory response to traumatic brain injury contribute to this process, however, their role remains largely undefined. We HYPOTHESIZE that neutrophils accumulate in brain and contribute to injury extension after traumatic brain injury. Related to this, we also propose studies to define changes in expression of adhesion receptors for neutrophils on the cerebrovascular endothelium after cerebral trauma, and to examine the effect of hypothermia on posttraumatic neutrophil accumulation and adhesion molecule expression. Our SPECIFIC AIMS are: 1) To quantitate and localize neutrophil accumulation (myeloperoxidase, immunohistochemistry, rhodamine-labeled neutrophils) and endothelial expression of neutrophil adhesion receptor (ICAM-1) in brain after controlled cortical contusion in rats, 2) To assess the effect of neutrophil depletion (mouse monoclonal anti-rat neutrophil antibody [RP-3]) on markers of secondary brain injury including hyperemia (autoradiography), edema (proton-density MRI), intracranial pressure (cisternal catheter), and lesion volume (T2- weighted MRI) after controlled cortical contusion in rats, 3) To define the contribution of the integrin and selectin adhesion pathways to posttraumatic neutrophil accumulation in brain, 4) To determine if transient (4h), moderate (32degreesC) hypothermia delays posttraumatic neutrophil accumulation and endothelial expression of adhesion molecules in brain, 5) To determine if more prolonged hypothermia (8 h or 12 h) produces a more sustained or permanent effect on posttraumatic neutrophil accumulation, and 6) To determine if transient, moderate cerebral hypothermia reduces secondary brain injury in our model. The cellular and molecular events in the acute inflammatory response are highly dependent on both the nature of the stimulus and the tissue involved. Trauma is a unique, complex, and important inflammatory stimulus, and the brain is a similarly unique tissue, particularly related to the highly differentiated endothelium (blood-brain barrier). Thus, to develop effective anti-neutrophil strategies targeting the acute inflammatory response to cerebral trauma, the specific cellular and molecular events involved must be defined. Recent developments in our ability to modify inflammation and in the clinical use of hypothermia make the proposed research timely and clinically relevant

[unreadable] DESCRIPTION (provided by applicant): In traumatic brain injury (TBI), adenosine activates high affinity A1 receptors conferring anti-excitotoxic effects. After TBI, however, adenosine levels are high-activating lower affinity A2a receptors that may down-regulate A1 and confer direct neurotoxicity. In models of Parkinson's disease, A2a receptor antagonists are neuroprotective. We reported neuroprotective effects of adenosine after TBI-via anti-excitotoxic effects at the A1 receptor. However, activation of lower affinity A2a receptors could negate this benefit. Out pilot studies show that A2a receptor ko mice are neuroprotected vs wt after experimental TBI and administration of the A2a agonist CGS21680 worsens outcome. However, A2a receptor agonists increase cerebral blood flow (CBF), a finding that must be reconciled. Our clinical studies show that increases in adenosine in cerebrospinal fluid (CSF) are associated with poor outcome. A therapeutic opportunity for A2a receptor antagonists is suggested; however, this pathway must be first studied in experimental TBI. A2a receptor signal transduction is coupled to adenyl cyclase (AC). We reported progressive increases in cAMP levels in CSF after clinical TBI. Hypothesis: Treatment with A2a receptor antagonists or inactivation of the A2a receptor will improve outcome after experimental TBI. Using the controlled cortical impact (CCI) model of TBI in mice and rats, we will address five aims: (1) Determine A2a receptor dynamics after CCI in mice and rats, (2) Assess the role of the A2a receptor in determining biochemical (glutamate, ACh, cAMP), functional, and histological outcome after CCI in mice and rats, including A2a receptor ko mice, (3) Assess the effects of A2a receptor activation on CBF and cerebral metabolic rate after CCI in rats. (4) Define the role of A2a receptor-mediated activation of AC after CCI in mice and rats, (5) Determine the role of the A1 receptor in the detrimental effects of A2a agonists in CCI using A1 receptor ko mice, and (6) To bridge bench and bedside after severe TBI in humans, using CSF samples from 161 patients, we will quantify levels of the non-selective adenosine receptor antagonist caffeine (and metabolites) to test the hypothesis that acute caffeine consumption is associated with favorable outcome and reduced cAMP. These studies explore the most promising adenosine-based therapy for TBI-A2a receptor antagonists. Our bench to bedside track record ensures translation to the clinic. [unreadable] [unreadable]

We propose to operate a core facility that performs the necessary procedures related to Animal Modeling and Outcome Assessment for the University of Pittsburgh Brain Trauma Research Center program project grant (PPG). The facilities in this core will be used by the following principal investigators (PIs) involved in the individual proposals in this PPG application: 1) Steven Graham, M.D., Ph.D., Project #1, "bcl-2 family genes in traumatic brain injury (TBI)", 2) Steven Dekosky., Project $2, "IL-1beta, APP metabolism and hypothermia in head injury", 3) Patrick Kochanek, M.D., Project #3, "iNOS and TBI", 4) Edward Dixon, Ph.D., Project #4 "Dopaminergic mechanisms of TBI," and 5) Robert Clark, M.D., Project #5 "PARS activation after TBI." The core facility will address the following Specific Aims 1) To provide a centralized facility for surgery and anesthesia for all of the proposed experimental manipulations of mice and rats within the PPG, 2) To provide uniform injury for all studies in mice and rats in the PPG (controlled cortical impact [CI] model, and CCI model with secondary insult) and ensure consistency by performing quality control analysis, 3) To provide a centralized facility for the standardized assessment of functional outcome parameters (motor and cognitive) within the PPG, 4) To provide a centralized facility for the standardized assessment of histological outcome parameters (contusion volume and hippocampal neuron counting) within the PPG, 5) To facilitate interaction between the PIs within the PPG, and facilitate interaction with collaborating investigators at the Pittsburgh NMR Center for Biomedical Research (Carnegie Mellon University), and 6) To provide a standardized hypothermia regimen for all studies using this therapy in rats in the PPG. The successful execution of these specific aims will provide for optimal resource utilization by the investigators include the use of 1) technician time, 2) laboratory space, 3) equipment, 4) supplies, 5) animals, and 6) Magnetic Resonance Imaging time. At the same time, quality control will be ensured for all of the studies in mice and rats, and a structured environment for interaction between the PIs within the PPG will be provided.

Inducible nitric oxide synthase (iNOS) is a NOS isoform that is involved in the inflammatory response. iNOS may govern key mechanisms in the evolution of injury, protection and repair, including vascular regulation, inflammation, cytoprotection, cytotoxicity, and regeneration. In the traumatic brain injury (TBI), iNOS is expressed in a variety of cell types in the peri-trauma region. We performed studies examining long-term outcome after TBI and observed a powerful endogenous neuroprotectant effect of iNOS in two species. Our studies are supported by an expanding body of literature revealing important beneficial effects of iNOS in response to injury inside and outside of the CNS. The hypothesis of this proposal is that iNOS is expressed that TBI and is a powerful endogenous neuroprotectant. Specific aims are as follows: 1) Determine the time course, magnitude, and cellular localization of iNOS induction after experimental TBI in both mice and rats, 2) Test whether iNOS is an endogenous neuroprotectant and improves both histopathological and functional outcome after TBI, using both iNOS KO mice and iNOS inhibitors in rats. Also investigate the possibility that there is a biphasic role of iNOS after TBI, with early detrimental effects, but beneficial effects overall, 3) Test in both mice and rats if over-expression of iNOS by gene transfer with adenovirus-based vector is neuroprotective after TBI, 4) Determine in our mouse and rat models how iNOS confers its neuroprotective effects, including evaluation of downstream mediators such as cytokines, nerve growth factor (NGF), and cerebral blood flow (CBF), and 5) Define, in humans with severe TBI, the global and local production of NO, as assessed by nitrite/nitrate levels in cerebrospinal fluid (CSF) and brain interstitial fluid, respectively, and determine the time course, magnitude, and cellular localization of iNOS induction in human cerebral contusions. Our established CCI models will be used to produce TBI in mice and rats. Expression of iNOS will be studied using RT-PCR, enzyme activity, and immunohistochemistry. Two inhibitors of iNOS (aminoguanidine and N6-(ininoethyl1)-L-lysine) and iNOS KO mice will be used. A replication deficient adenovirus that expresses human iNOS will be used to transfect brain regions in vivo, both before and after injury. Outcome evaluation will include motor and cognitive (Morris water maze) tasks, histopathology, CBF (perfusion NMR), cytokines and NGF (ELISA), and macrophage/lymphocyte infiltration (by immunohistochemistry and flow cytometric analysis). In humans with severe TBI, nitrite/nitrate levels will be used as a marker of NO in both CSF and brain interstitial fluid (microdialysis). Brain samples from patients undergoing emergency resection of contusion, will be studied using immunohistochemistry. Confirming that iNOS is a neuroprotectant, showing that over-expression of iNOS as beneficial, and defining the mechanisms involved in this effect are key steps toward the of a novel treatment. Finally, these studies will unite bench to bedside for this important mechanism in TBI.

DESCRIPTION (provided by applicant): The University of Pittsburgh Brain Trauma Research Center has been investigating the molecular and cellular mechanisms of secondary brain injury (physiologic and neurochemical responses of the injured brain) since its inception in 1991. Our studies have lead to an improved understanding of specific molecular mechanisms likely to be responsible for early and delayed neurologic dysfunction following TBI. The investigators of the Center have resulted in more than 188 peer-reviewed journal articles and book chapters during the last five years. TBI initiates pathological biochemical cascades that can persist long after survival. An increased understanding of the mechanisms of these cascades and their attenuation by translatable therapies are the primary scientific goals of our program project. The specific projects selected for this proposal represent a logical extension of the research conducted by primary investigators of the previous program project grant, as well as a new area of exploration introduced by Dr. C. Edward Dixon. These include the study of (1) nitrosative stress and PARP activation, (2) statins therapies and their interaction with Ap in cell death, (3) effects of calcineurin inhibition on neuronal death and plasticity, (4) Fas-mediated cell death, and (5) mechanism(s) underlying the endogenous beneficial effects of iNOS. All of the projects include clinically relevant time points, translatable treatments, and at least one specific aim that test the relevance of the basic science hypotheses to TBI in humans. Because of this we will be able to correlate the findings of our primary investigations with human TBI and determine their relative importance in effecting neurologic outcome. In this way, the completion of our specific aims can be expected to define critical acute and chronic molecular mechanisms of secondary brain injury and identify treatments most likely to be beneficial to TBI patients.

DESCRIPTION (provided by applicant): The term "cAMP" is used universally to refer to 3',5'-cyclic adenosine monophosphate (3',5'- cAMP), the famous "second messenger" discovered by Dr. Earl Southerland. Importantly, the vast majority of investigators measure 3',5'-cAMP using various commercially available assay kits, and our group is the ONLY group in the world that routinely measures 3',5'-cAMP using high performance liquid chromatography-tandem mass spectrometry(LC-MS/MS). Dr. Jackson's lab focuses mostly on the role of purines in renal physiology/pharmacology, and routinely employs LC-MS/MS, rather than assay kits, to measure 3',5'-cAMP. In experiments unrelated to the brain, Dr. Jackson's lab serendipitously discovered that the kidney produces more 2',3'-cAMP (a positional isomer of 3',5'- cAMP) than 3',5'-cAMP. Dr. Kochanek's lab focuses primarily on mechanisms of traumatic brain injury (TBI), and has discovered that purines play a major role in protecting the brain from TBI. Because of Dr. Jackson's expertise in measuring purines and Dr. Kochanek's interest in the role of purines in TBI, a natural and productive collaboration evolved between these investigators. After the serendipitous discovery of 2',3'-cAMP in kidneys, Drs. Jackson and Kochanek decided to investigate whether 2',3'-cAMP exists in the brain. In preliminary studies, LC-MS/MS analysis of 44 samples of cerebral spinal fluid (CSF) from TBI patients showed that 2',3'-cAMP is a major constituent of "cAMP" in CSF from patients with TBI. In addition to 2',3'-cAMP, the levels of 2'-AMP, adenosine and inosine (metabolite of adenosine) were also measured in these same samples. Importantly, there was an large and significant correlation between 2',3'-cAMP and 2'-AMP, 2'-AMP and adenosine and 2'-AMP and inosine in human CSF. These surprising discoveries suggested a critical need to explore the role of 2',3'-cAMP in TBI pathophysiology, particularly with respect to the role of 2',3'-cAMP as a precursor for adenosine. Accordingly, the purpose of this exploratory project is to begin to characterize the significance of 2',3'-cAMP in TBI by testing the innovative concept that 2',3'-cAMP is involved in a "CNPase Neuroprotection Mechanism" in which brain injury leads to release of 2',3'-cAMP from mRNA, and 2',3'-cAMP is then metabolized to 2'-AMP by CNPase followed by conversion of 2'-AMP to adenosine. PUBLIC HEALTH RELEVANCE: TBI is a major source of mortality, morbidity and life-long impairment in both the civilian and military populations, with limited treatment options and generally dismal outcomes. The CNPase Neuroprotective Mechanism could be extremely important in producing extracellular adenosine during TBI, thus providing this protective, "retaliatory" metabolite to mitigate cellular damage. Manipulation of this mechanism by drugs could provide novel approaches to prevent and treat TBI.

DESCRIPTION (provided by applicant): The term cAMP usually refers to the second messenger 3',5'-cyclic adenosine monophosphate. We serendipitously discovered that organ systems can produce (from mRNA degradation) and export to the extracellular compartment a positional isomer of 3',5'-cAMP, namely 2',3'-cAMP. We showed that organ systems convert extracellular 2',3'-cAMP to 2'-AMP + 3'-AMP and can metabolize 2'-AMP and 3'-AMP to adenosine. We refer to this pathway as the 2',3'-cAMP-adenosine pathway. We also showed that extracellular 2',3'-cAMP increases greatly post-traumatic brain injury (TBI) in brain in rodents and humans; and that when the pathway is impaired, TBI outcomes worsen in rodents. Intracellular 2',3'-cAMP opens mitochondrial permeability transition pores while extracellular adenosine is neuroprotective. Thus the 2',3'- cAMP-adenosine pathway may be important in TBI because it eliminates an intracellular neurotoxin (export of 2',3'-cAMP) and generates an extracellular neuroprotectant (conversion of 2',3'-cAMP to adenosine). We also identified the enigmatic myelin protein 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) to be the major enzyme that metabolizes extracellular 2',3'-cAMP to 2'-AMP (a key step toward conversion into adenosine). KO mice lacking CNPase produce less extracellular adenosine post-TBI, are more susceptible to injury and develop axonal degeneration with age despite no gross myelin abnormalities. Hypothesis: the 2',3'-cAMP- adenosine pathway is an endogenous cytoprotective mechanism after TBI. We will elucidate which CNS cell types produce 2',3'-cAMP, what kinds of injury trigger 2',3'-cAMP production, how 2',3'-cAMP is transported out of cells, how downstream AMPs are converted to adenosine, and if manipulating the 2',3'-cAMP-adenosine pathway alters secondary damage. Specific Aim 1: To determine which CNS cell types produce 2',3'-cAMP after injury. Because in vivo TBI increases extracellular 2',3'-cAMP, it is important to determine which CNS cells produce 2',3'-cAMP and whether the effect is injury-type dependent. Aim 1 will determine if metabolic stress, hypoxia or mechanical injury enhances 2',3'-cAMP production by astrocytes, microglia, neurons or oligodendrocytes. Specific Aim 2: To determine whether Multidrug Resistance Protein 4 (MRP4) mediates egress of 2',3'-cAMP. Because 2',3'-cAMP is an intracellular toxin, it is critical to elucidate how 2',3'-cAMP is extrude from CNS cells. Aim 2 will test the hypothesis that MRP4 exports 2',3'-cAMP. Specific Aim 3: To determine if Tissue Alkaline Phosphatase (TAP) participates in the extracellular metabolism of 2'-AMP and 3'- AMP (downstream metabolites of 2',3'-cAMP) to adenosine. Because extracellular adenosine is neuroprotective it is essential to understand how extracellular 2'-AMP and 3'-AMP are converted to extracellular adenosine. Specific Aim 4: To test the hypothesis that the 2',3'-cAMP-adenosine pathway is an endogenous protective mechanism post-TBI. Aim 4 will further test the hypothesis that the 2',3'-cAMP- adenosine pathway is cytoprotective by determining the effect of inhibiting or augmenting it on TBI outcomes.