Allison Kunze

The immunologic profile of adoptively transferred lymphocytes influences stroke outcome of recipients

Animals that have myelin basic protein (MBP) specific lymphocytes with a Th1(+) phenotype have worse stroke outcome than those that do not. Whether these MBP specific cells contribute to worsened outcome or are merely a consequence of worse outcome is unclear. In these experiments, lymphocytes were obtained from donor animals one month after stroke and transferred to naïve recipient animals at the time of cerebral ischemia. The MBP specific phenotype of donor cells was determined prior to transfer. Animals that received either MBP specific Th1(+) or Th17(+) cells experienced worse neurological outcome, and the degree of impairment correlated with the robustness of MBP specific Th1(+) and Th17(+) responses. These data demonstrate that the immunologic phenotype of antigen specific lymphocytes influences stroke outcome.

Promiscuity of autoimmune responses to MBP after stroke

In this study we examined Th1 and Th17 immune responses to rat myelin basic protein (MBP), bovine MBP, human MBP, MBP 68-86, MBP 63-81 and ovalbumin in Lewis rats to determine which MBP antigen is recognized following ischemic brain injury. Responses were compared to animals immunized to rat MBP. Data show that immune responses following immunization with rat MBP are promiscuous with cross reaction to MBP from other species. After stroke, few animals develop Th1 or Th17 responses to MBP, but when those responses occur, especially Th1 responses to rat MBP in the brain, they are predictive of worse stroke outcome.

Anamnestic Recall of Stroke-Related Deficits. An Animal Model

Background and Purpose— Anamnestic recall of stroke-related deficits is a common clinical observation, especially during periods of systemic infection. The pathophysiology of this transient re-emergence of neurological dysfunction is unknown. Methods— Male Lewis rats underwent 3 hours middle cerebral artery occlusion and were treated with lipopolysaccharide or saline at the time of reperfusion. The delayed-type hypersensitivity (DTH) response to myelin basic protein was examined 28 days after middle cerebral artery occlusion. Changes in behavioral outcomes were assessed after DTH testing and repeat administration of lipopolysaccharide or saline at 34 days. At the time of euthanasia (36 days), the immunologic response of splenocytes to myelin basic protein, neuron-specific enolase, and proteolipid protein was determined by enzyme-linked immunospot assay and the number of lymphocytes in the brain determined by immunocytochemistry. Results— Animals treated with lipopolysaccharide at middle cerebral artery occlusion had a greater DTH response to myelin basic protein than animals treated with saline. Among those animals that had fully recovered on a given behavioral test before DTH testing, those treated with lipopolysaccharide at middle cerebral artery occlusion displayed more neurological deterioration after DTH testing and had more CD8+ lymphocytes within the ischemic core of the brain. Furthermore, the Th1 immune response to brain antigens in the spleen was more robust among those animals that deteriorated after DTH testing and there were more CD4+ lymphocytes in the penumbral region of animals with a Th1 response to myelin basic protein. Conclusions— Our data suggest that an immune response to the brain contributes to the phenomenon of anamnestic recall of stroke-related deficits after an infection. The contribution of the immune response to this phenomenon deserves further investigation.

Effect of Antibiotic Class on Stroke Outcome

Background and Purpose— Infections are common after stroke and associated with worse outcome. Clinical trials evaluating the benefit of prophylactic antibiotics have produced mixed results. This study explores the possibility that antibiotics of different classes may differentially affect stroke outcome. Methods— Lewis rats were subjected to transient cerebral ischemia (2 hours) and survived for 1 month. The day after stroke they were randomized to therapy with ceftiofur (a &bgr;-lactam antibiotic), enrofloxacin (a fluoroquinolone antibiotic), or vehicle (as controls) and underwent the equivalent of 7 days of treatment. Behavioral tests were performed weekly until euthanization. In a subset of animals, histology was done. Results— There were no differences in outcomes at 24 hours or 1 week after stroke among the different groups. At 1 month after stroke, however, performance on the rotarod was worse in enrofloxacin-treated animals when compared with control animals. Conclusions— Independent of infection, the antibiotic enrofloxacin was associated with worse stroke outcome. These data echo the clinical observations to date and suggest that the secondary effects of antibiotics on stroke outcome should be considered when treating infection in subjects with stroke. The mechanism by which this antibiotic affects outcome needs to be elucidated.

Peroxiredoxin 5 (PRX5) Is Correlated Inversely to Systemic Markers of Inflammation in Acute Stroke

Background and Purpose— Peroxiredoxins are endogenous antioxidants that function as peroxide and peroxynitrite scavengers. Extracellular peroxiredoxins, however, are shown to initiate inflammation within the ischemic brain through activation of Toll-like receptors. Based on this observation, we hypothesized that plasma peroxiredoxin concentrations in ischemic stroke would correlate biomarkers of inflammation and predict poor outcome. Methods— In a prospective study of patients with ischemic stroke, plasma peroxiredoxin 5 (PRX5) concentrations and inflammatory biomarkers at day 3 after stroke onset were correlated and the association between PRX5 at day 3 and outcome at 3 months assessed. Results— PRX5 concentrations were available for 98 patients and were lower in those with more severe strokes (P=0.001). PRX5 was inversely correlated to biomarkers of inflammation at day 3 after stroke and did not predict 3-month outcome. Conclusions— Plasma PRX5 is decreased in severe stoke and inversely correlated to biomarkers of systemic inflammation. These data suggest that PRX5 is not a proinflammatory mediator in acute stroke. Moreover, the inverse relationship between PRX5 and stroke severity suggests that PRX5 is either consumed or its production is impaired in severe stroke. Further study is needed to define the potential role of PRX5 in stroke.

The contribution of antibiotics, pneumonia and the immune response to stroke outcome

BACKGROUND Infections are common following stroke and associated with worse outcome. Using an animal model of pneumonia, we assessed the effect of infection and its treatment on the immune response and stroke outcome. METHODS Lewis rats were subjected to transient cerebral ischemia and survived for 4weeks. One day after stroke animals were exposed to aerosolized Staphylococcus aureus, Pseudomonas aeruginosa or saline. Antibiotics (ceftiofur or enrofloxacin) were started immediately after exposure or delayed for 3days. Behavioral tests were performed weekly. ELISPOT assays were done on lymphocytes from spleen and brain to assess autoimmune responses to myelin basic protein (MBP). RESULTS Among animals that received immediate antibiotic therapy, infection was associated with worse outcome in ceftiofur but not enrofloxacin treated animals. (The outcome with immediate enrofloxacin therapy was so impaired that further worsening may have been difficult to detect.) A delay in antibiotic therapy was associated with better outcomes in both ceftiofur and enrofloxacin treated animals. Infection was associated with an increased likelihood of developing Th1(+) responses to MBP in non-infarcted brain (OR=2.94 [1.07, 8.12]; P=0.04), and Th1(+) responses to MBP in spleen and non-infarcted brain were independently associated with a decreased likelihood of stroke recovery (OR=0.16 [0.05, 0.51; P=0.002 and OR=0.32 [0.12, 0.84]; P=0.02, respectively). CONCLUSIONS Infection worsens stroke outcome in ceftiofur treated animals and increases Th1 responses to MBP. These data may help explain how infection worsens stroke outcome and suggest that treatment of infection may contribute to this outcome.

Chemical Sympathectomy, but not Adrenergic Blockade, Improves Stroke Outcome

BACKGROUND A robust adrenergic response following stroke impairs lymphocyte function, which may prevent the development of autoimmune responses to brain antigens. We tested whether inhibition of the sympathetic response after stroke would increase the propensity for developing autoimmune responses to brain antigens. METHODS Male Lewis rats were treated with 6-hydroxydopamine (OHDA) prior to middle cerebral artery occlusion (MCAO), labetalol after MCAO, or appropriate controls. Behavior was assessed weekly and animals survived to 1 month at which time ELISPOT assays were done on lymphocytes from spleen and brain to determine the Th1 and Th17 responses to myelin basic protein (MBP), ovalbumin (OVA), and concanavalin A. A subset of animals was sacrificed 72 hours after MCAO for evaluation of infarct volume and lymphocyte responsiveness. Plasma C-reactive protein (CRP) was measured as a biomarker of systemic inflammation. RESULTS Despite similar initial stroke severity and infarct volumes, 6-OHDA-treated animals lost less weight and experienced less hyperthermia after stroke. 6-OHDA-treated animals also had decreased CRP in circulation early after stroke and experienced better neurological outcomes at 1 month. The Th1 and Th17 responses to MBP did not differ among treatment groups at 1 month, but the Th1 response to OVA in spleen was more robust in labetalol and less robust in 6-OHDA-treated animals. CONCLUSIONS Chemical sympathectomy with 6-OHDA, but not treatment with labetalol, decreased systemic markers of inflammation early after stroke and improved long-term outcome. An increase in Th1 and Th17 responses to MBP was not seen with inhibition of the sympathetic response.