Inflammation, caffeine and adenosine in neonatal hypoxic ischemic brain injury

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

Long Lasting Local and Systemic Inflammation after Cerebral Hypoxic ischemia in Newborn Mice

Background: Hypoxic ischemia (HI) is an important cause of neonatal brain injury and subsequent inflammation affects neurological outcome. In this study we performed investigations of systemic and local activation states of inflammatory cells from innate and adaptive immunity at different time points after neonatal HI brain injury in mice. Methodology/Principal Findings: We developed a multiplex flow cytometry based method combined with immunohistochemistry to investigate cellular immune responses in the brain 24 h to 7 months after HI brain injury. In addition, functional studies of ex vivo splenocytes after cerebral hypoxic ischemia were performed. Both central and peripheral activation of CD11b + and CD11c + antigen presenting cells were seen with expression of the costimulatory molecule CD86 and MHC-II, indicating active antigen presentation in the damaged hemisphere and in the spleen. After one week, naïve CD45rb + T-lymphocytes were demonstrated in the damaged brain hemisphere. In a second phase after three months, pronounced activation of CD45rb 2 T-lymphocytes expressing CD69 and CD25 was seen in the damaged hemisphere. Brain homogenate induced proliferation in splenocytes after HI but not in controls. Conclusions/Significance: Our findings demonstrate activation of both local and systemic immune responses months after hypoxic ischemic neonatal brain injury. The long term immune activation observed is of general importance for future studies of the inflammatory response after brain injury as most previous studies have focused on the first few weeks afte

Single dose caffeine protects the neonatal mouse brain against hypoxia ischemia - Flow cytometric data

Caffeine 5mg/kg or phosphate buffered saline given directly after sham operation or brain hypoxia ischemia. Flow cytometric evaluation of brain immune cell content 24h, 72h and 2 weeks after brain injury. <br

Systemic inflammatory cell response after hypoxic ischemic (HI) brain injury.

<p>(A) CD11b⁺ cells per 10⁶ live cells in the spleen over time after HI brain injury (left) and the CD86⁺ proportion of these cells (right). (B) CD11c⁺ cells per 10⁶ live cells (left) and the CD86⁺ proportion of these cells (right). (C) CD4⁺ and CD8⁺ T-lymphocytes expressing the activation markers CD69 and CD25 in spleen. Red circle = HI, green diamond = sham operated, and blue square = unoperated.</p

Local and peripheral dynamics of innate and adaptive immune activation after hypoxic ischemic brain injury.

<p>(A) Temporal correlation of activated cells after HI brain injury. Note the 24 h and one week activation of CD11b⁺ cells as well as brain T-cell infiltration after one week. Black filled line; activated CD11b⁺CD86⁺ cells in the brain. Green short dashed line; activated CD8⁺CD69⁺ and blue long dashed line; CD4⁺CD69⁺ T-lymphocytes in the brain. Red dotted line; activated splenetic T-lymphocytes. Yellow long dashed line; Proportion CD86 expressing splenetic APCs. (B) Schematic overview of cellular inflammation after HI brain injury.</p

Single Dose Caffeine Protects the Neonatal Mouse Brain against Hypoxia Ischemia

<div><p>In this randomized blinded study, we investigated caffeine 5 mg/kg treatment given directly after neonatal brain hypoxia ischemia. Brain morphology, behavior and key brain infiltrating immune populations were examined. Caffeine treatment significantly improves outcome when compared to phosphate buffered saline. Flow cytometric analysis of immune responses revealed no persistent immunological alterations. Given its safety caffeine emerges as a candidate for neuroprotective intervention after neonatal brain injury.</p></div

Heat map of all investigated immune populations after sham operation or HI 24h, 72h and two weeks after randomization to caffeine or PBS (n = 3–5 in each group, total n = 52).

<p>The data was normalized to zero for the lowest value and one for the highest value in each variable with zeros in blue through yellow to ones values in red. Whites are missing values. MFI = Median florescence intensity.</p

Splenocyte proliferation and VCAM-1 expression in brain after hypoxic ischemic brain injury.

<p>(A) Proliferation index of splenocytes from mice three and five months after HI or sham operation measured by [³H] Thymidine incorporation after stimulation with brain extract. (B) VCAM-1 expression in the infarcted hippocampus region (star) and blood vessels (arrow) as visualized by immunohistochemistry. Ruler indicates 500 µm. (C) VCAM-1 expression in damaged (squares) and undamaged (circles) hemispheres over time after HI brain injury.</p

Presence of double-positive CD4⁺CD8⁺ T-cells after hypoxic ischemic brain injury.

<p>(A) CD4⁺ and CD8⁺ T-lymphocytes in unoperated age matched control (left) and damaged brain hemisphere (right) two weeks after HI. Note the double positive population marked in red. (B) Proportion CD4⁺CD8⁺ double positive T-lymphocytes in brain and (C) spleen after HI. Red circle = infarcted hemisphere (B) or HI (C), black triangle = uninjured hemisphere, blue diamond = sham operated, and green square = unoperated.</p

Local innate inflammatory cell response after hypoxic ischemic (HI) brain injury.

<p>(A) Time-line with experimental setup. (B) Representative cross-section of MAP-2 (live neurons) stained mouse brain two weeks after brain damage. (C) Number of CD11b⁺ (left) and CD11c⁺ cells (right) expressing the activation marker CD86 over time. (D) Number of CD11b⁺ (left) and CD11c⁺ cells (right) expressing MHC-II (H2IAb) over time. Red circle = infarcted hemisphere, black triangle = uninjured hemisphere, blue diamond = sham operated, and green square = unoperated. * = p<0.05, ** = p<0.01 and *** = p<0.001. Compared to sham and unoperated controls. Error bars represent SEM.</p