Inflammation, caffeine and adenosine in neonatal hypoxic ischemic brain injury

Sammanfattning: Background: Brain injury during the neonatal period has potentially lifelong consequences for a child. Perinatal infections and inflammation can induce preterm birth and unfavorable cognitive development, Thus inflammation has received enthusiastic interest for potential therapeutic approaches seeking to protect the newborn brain. Experimental evidence demonstrates that inflammation induces brain injury succeeding the initial insult. A key cytokine in brain injury is the tumor necrosis factor (TNF-α), with reported detrimental cytotoxic effects on selected neuronal populations. Nonetheless, important functions of TNF-α in cerebral homeostasis and development have also been described. Caffeine is used against apneas of prematurity, with noticeable protection against cognitive delay and cerebral palsy. The main effects of caffeine at clinically relevant doses are mediated through inhibition of adenosine receptors. Adenosine is formed from adenosine triphosphate, the main transporter of chemical energy in the cell, which is readily cleaved to adenosine upon extracellular release or extensive leakage from injured necrotic cells. Hence, adenosine signaling is tightly interrelated with local energy levels and cell injury. In addition, adenosine modulates inflammatory responses in profound ways. Methods: In mouse models of premature excitotoxic lesions and full term hypoxic ischemic brain injury we investigated blockade of TNF-α with and without interleukin IL-1 or lipopolysaccharide (LPS) induced systemic inflammation. Furthermore, in the hypoxic ischemic model we developed a flow cytometry based method to investigate temporal distribution of brain infiltrating and splenetic immune cells and their activation. To analyze the data in an unbiased way, we next adapted a data driven gating methodology. Moreover, we used principal component analysis to discriminate between experimentally entangled variables. Utilizing these techniques, we explored the effect of genetic inactivation of adenosine A1 and A2A receptors in the hypoxic ischemic model. We also tested the unselective, competitive adenosine receptor antagonist caffeine and assessed the effect on outcome and immune activation. Results: Blockade of TNF-α protected the brain against excitotoxic lesions in the presence but not absence of systemic inflammation. No protection was observed in the full term hypoxic ischemic model. Persistent lymphocyte activation was found three months after the lesion. Moreover, spleenocytes harvested five months after neonatal brain damage proliferated when stimulated with brain homogenate in contrast to sham operated counterparts. Adenosine A1 receptor deficient mice acquired significantly larger infarcts and associated adverse behavioral outcome compared to wild type. There were specific alterations in the immune responses induced after brain injury, including impaired cytotoxic function and dysregulation of regulatory B-lymphocytes. Adenosine A2A receptor knockout mice developed increased atrophy compared to wild type after hypoxic ischemia, an effect accompanied by functional deficits in behavioral tests. Furthermore, a compensated functional insufficiency was estimated in the regulatory T- lymphocyte compartment in combination with a seemingly inadequate number of myeloid derived suppressor like cells, accompanied by a reversed, increased response in innate antigen presenting cells in the knockout. Finally, we report neuroprotective properties of 5 mg/kg caffeine given directly after neonatal brain injury. Discussion: TNF-α blockade could potentially protect against preterm excitotoxic brain injury. Only patients with concurrent systemic inflammation would potentially benefit. Moreover, concern about adverse effects exists, why TNF-α blockade for neonatal brain injury is likely not clinically applicable in the near future. Persistent long term cerebral adaptive immune activation, preceded by systemic immune activation in spleen was discovered. Remarkably, spleenocytes from animals subjected to brain injury responded to brain antigen five months after brain damage, whereas spleenocytes from uninjured did not, suggesting formation of immunological memory that might affect long term outcome and provoke autoimmunity later in life. To avoid bias from manual gating of flow cytometry data we developed a data driven approach adapted for brain infiltrating immune cells. Furthermore, we deployed principal component analysis to verify biological relevance in the pattern of immune activation and to discriminate between genotype and injury size effects, since they are experimentally inseparable. Thus we could predict genotype and whether they acquired brain injury or not, from the flow cytometric immune activation pattern alone. Adenosine A1 receptor deficient mice display signs of regulatory B-lymphocyte dysfunction that imply a novel adenosinergic mechanism of B-lymphocyte regulation. In addition, these animals displayed signs of altered cellular cytotoxic immunity. Thus considerable effects on immune activation were present in the A1 receptor knockouts compared to wild type, adding another mechanism linked to worse outcome after hypoxic ischemic brain injury in these animals. Deletion of the adenosine A2A receptor similarly causes worse outcome, however, the alteration of the immune response is completely different. Fundamental changes were observed in regulatory populations like monocyte derived suppressor like cells and regulatory T-lymphocytes. Extensive activation of cytotoxic populations in the adenosine A2A receptor knockout links insufficient regulatory immune function with adverse behavioral and morphological outcome. We also propose a novel hypothesis that short term blockade of adenosine A2A receptors offers neuroprotection whereas long term blockade is detrimental by immunological mechanisms. Thus we tested the tentative therapeutic potential of caffeine, an unselective competitive antagonist of adenosine receptors. Caffeine 5mg/kg given directly after the insult resulted in reduced injury size after neonatal hypoxic ischemia. Since caffeine is a relatively well studied substance with negligible adverse long term effect in technically sound studies absent of significant bias, this approach has a clear clinical relevance. Are we ready for a clinical trial?