Intracerebral hemorrhage (ICH) is a type of stroke caused by bleeding into the brain, resulting in significant death and disability. Secondary and delayed pathophysiological events can contribute to the death and disability of ICH patients. These events are characterized by a breakdown of the blood-brain barrier, vasogenic edema development and cell death in white and gray matter. Investigators on this proposal have recently discovered that, following hemorrhage in the brain, a new molecular species can be produced by the oxidation of bilirubin. These bilirubin oxidation products (BOXes) are cytotoxic and preliminary data are presented in the proposal indicating that they can contribute to pathological events observed following ICH. This proposal attempts to investigate secondary pathologic mechanisms following ICH to determine whether BOXes play a role in ICH induced brain tissue injury. The central hypothesis is that BOXes underlie vasogenic edema formation and white and gray matter injury following ICH. The proposal employs a porcine model producing ICH by injection of autologous blood.
Aim 1 proposes to measure the concentration of BOXes in the hematoma and perihematomal brain tissue.
Aim 2 will add BOXes to the hematoma and then assess the damage and examine the underlying pathology observed following ICH.
Aim 3 proposes to investigate potential therapeutic strategies designed to decrease BOXes. ? ? SIGNIFICANCE: As the investigators point out, ICH represents about 10% of all strokes and has the highest mortality rate and poorest prognosis in survivors. Improved understanding of the pathobiology of ICH and development of new therapeutic approaches would represent a significant medical advance. In addition, the potential contribution of oxidized bilirubin to ICH pathology is an area that has not been widely explored. However, fundamental flaws in the proposed experimental approaches significantly compromise the interpretability and the utility of data derived from this research (see below). ? ? APPROACH: The approach of the investigators does include some strong points. The Principal Investigator and his colleagues have significant experience in studying cerebrovascular events associated with trauma and ischemia. In addition, they have developed a porcine model of ICH which is the central component of the proposed research. A number of aspects of this model including the presence of edema and brain damage have been investigated in preliminary experiments. Thus, there are pilot data to support some components of this proposal. ? ? The central problem confronting this proposal is that it does not provide rigorous mechanisms for testing the central hypothesis that BOXes underlie vasogenic edema formation and white and gray matter injury following ICH. In general, the proposal is not well organized and excessive amounts of data are provided on subarachnoid hemorrhage (SAH) that is not directly relevant to this proposal. For example, substantial amounts of pilot data are reviewed discussing the potential role of BOXes in vasospasm, an end point not measured in this proposal and not presumably an important component of ICH. There is no systematic provision to understand precisely how BOXes could, in fact, effect vascular smooth muscle to produce blood-brain barrier opening or to understand what mechanisms are actually involved in white and gray matter injury. Presumably, these could be direct cellular mechanisms unrelated to vascular events, but this point is never clearly addressed in the proposal. ? ? The first Specific Aim proposes to examine whether BOXes are found in the hematoma and brain tissue following ICH. Curiously, no pilot data are presented on measurement of BOXes in this model, only a time course for bilirubin (Fig. 18B, p. 41). In addition, the investigators propose to examine white and gray matter heme oxygenase-1 (HO-1) gene expression by quantitative RT-PCR and HO-1 protein expression by Western blotting and cellular localization by immunohistochemistry. It is not clear how these data on HO-1 will be integrated into studies of bilirubin and bilirubin oxidation. Since the central hypothesis is that both increased levels of bilirubin and oxidative stress are necessary to produce pathological levels of BOXes, it is curious that investigators have not chosen to study these relationships with more detail in this specific aim. ? ? Specific Aim 2 proposes to examine whether BOXes exacerbate ICH induced increases in blood-brain barrier permeability, vasogenic edema and development of white and gray matter injury in a dose-dependent fashion. The investigators propose to include BOXes along with infused blood to induce and exacerbate the ICH. Evans blue studies will assess blood-brain barrier permeability and vasogenic edema. White and gray matter injury will be measured by a standard histological and immunocytochemical methods. The investigators have presented pilot data that BOXes can exacerbate edema as well as white and gray matter injury. However, these studies were largely qualitative, and it is not clear how investigators will adequately quantify the end points to detect dose-dependent changes, especially given the small N's (5 per group) proposed in this study. In addition, the design represents a failure in clarity of hypothesis structure. There is considerable ambiguity about the investigators' views on primary and intermediate mechanisms of cell death. Are the mediating events of injury by BOXes related to changes in the vasculature, or are they direct effects of BOXes on brain cells? In any case, what are the mechanisms of cell death accompanying injury and what cell types are preferentially affected? None of these questions are addressed with any rigor in Specific Aim 2. ? ? Specific Aim 3 proposes to address the effects of therapeutic intervention on vasogenic edema development and brain injury following ICH. As most investigators recognize, even in animal models, treatment approaches can be extremely problematic, and no preliminary data are presented. This is an especially significant concern given the ambiguity and potentially qualitative nature of the multiple end points the investigators propose to assess and the relatively small number of animals utilized. The proposal to treat animals with antibodies to BOXes is especially problematic. The investigators recognize that BOXes antibodies can work only after BOXes are produced, so that some damage is likely. However, the larger problem seems to be identification of an optimal monoclonal or polyclonal antibody for pig. While they comment that they will conduct """"""""trial and error testing"""""""" with commercially available antibodies from other species, there is an extremely unsatisfying level of detail regarding this component of the proposal. ? ? INNOVATION: As mentioned above, the possibility that BOXes mediate ICH pathobiology is a relatively unexplored area. However, the techniques and approaches are not novel and often insufficient to address hypotheses rigorously. ? ? INVESTIGATOR: Dr. Clark is extremely experienced in cerebrovascular injury and is well qualified to conduct this research. In addition, he is assisted by other experienced investigators and technicians. ? ? ENVIRONMENT: The environment is adequate. The University of Cincinnati has an established history in studying cerebrovascular injury. ? ? OVERALL EVALUATION: This proposal, written by an experienced investigator, addresses an understudied hypothesis of the potential role of BOXes in the pathology of ICH. However, fundamental flaws in the experimental approaches proposed, as well as an absence of critical pilot significantly erode enthusiasm for this research. The investigator is strongly encouraged to develop a more focused proposal, directly addressing the potential mechanisms of bilirubin oxidation in CNS injury after ICH. ? ? ANIMAL WELFARE: No concerns. ? ? BIOHAZARDS: No concerns. ? ? BUDGET: Potentially some overlap with a funded project on SAH (a significant amount of pilot data included in this proposal). Not clear that all personnel are required. ? ?
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