Diet composition and sterilization modifies intestinal microbiome diversity and burden of Theiler's virus infection-induced acute seizures.

OBJECTIVE: Brain infection with Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6J mice can induce acquired epileptogenesis. Diet alters acute seizure incidence in TMEV-infected mice; yet it is unclear whether intestinal dysbiosis may also impact acute or chronic behavioral comorbidities. This study thus assessed the impact of diet formulation and sterilization on acute seizure presentation, gut microbiome composition, and epilepsy-related chronic behavioral comorbidities. METHODS: Baseline fecal samples were collected from male C57BL/6J mice (4- to 5-weeks-old; Jackson Labs) upon facility arrival. Mice were randomized to either autoclaved (AC) or irradiated diet (IR) (Prolab RMH 3000) or IR (Picolab 5053). Three days later, mice underwent intracerebral TMEV or phosphate-buffered saline (PBS) injection. Fecal samples were collected from a subset of mice at infection (Day 0) and Day 7 post-infection. Epilepsy-related working memory deficits and seizure threshold were assessed 6 weeks post-infection. Gut microbiome diversity was determined by 16S rRNA amplicon sequencing of fecal samples. RESULTS: TMEV-infected mice displayed acute handling-induced seizures, regardless of diet: 28 of 57 IR Picolab 5053 (49.1%), 30 of 41 IR Prolab RMH 3000 (73.2%), and 47 of 77 AC Prolab RMH 3000 (61%) mice displayed seizures. The number of observed seizures differed significantly by diet: IR Picolab 5053 diet-fed mice had 2.2 ± 2.8 seizures (mean ± standard deviation), IR Prolab RMH 3000 diet-fed mice had 3.5 ± 2.9 seizures, and AC Prolab RMH 3000 diet-fed mice had 4.4 ± 3.8 seizures during the 7-day monitoring period. Gut microbiome composition differed significantly in TMEV-infected mice fed the AC Prolab RMH 3000 diet, with measured differences in gram-positive bacteria. These mice also displayed worsened long-term working memory deficits. SIGNIFICANCE: Diet-induced differences in intestinal dysbiosis in the TMEV model are associated with marked changes in acute seizure presentation, symptomatic recovery, and onset of chronic behavioral comorbidities of epilepsy. Our study reveals a novel disease-modifying impact of dietary manipulation on intestinal bacterial species after TMEV-induced acute seizures.

Diet composition and sterilization modifies intestinal microbiome diversity and burden of Theiler's virus infection-induced acute seizures.

Objective: Central nervous system infection with Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6J mice can model acquired epileptogenesis. Diet alters the acute seizure incidence in TMEV-infected mice; yet it is unclear whether intestinal dysbiosis may also impact acute or chronic behavioral comorbidities. This study thus assessed the impact of diet sterilization in a specific pathogen-free vivarium on acute seizure presentation, the composition of the gut microbiome, and chronic behavioral comorbidities of epilepsy. Methods: Baseline fecal samples were collected from male C57BL/6J mice (4-5 weeks-old; Jackson Labs) upon arrival. Mice were randomized to either autoclaved (AC) or irradiated (IR) diet (Prolab RMH 3000 - UU diets) or IR (Picolab 5053 - UW IR diet). Mice then underwent intracerebral TMEV or PBS injection three days later. Fecal samples were collected from a subset of mice at infection (Day 0) and Day 7 post-infection. Epilepsy-related working memory deficits and seizure threshold were assessed 6 weeks post-infection. Gut microbiome diversity was determined by 16S rRNA amplicon sequencing of fecal samples. Results: TMEV-infected mice displayed acute handling-induced seizures, regardless of diet: 28/57 UW IR (49.1%), 30/41 UU IR (73.2%), and 47/77 UU AC (61%) mice displayed seizures. The number of observed seizures significantly differed: UW IR mice had 2.2±2.8 seizures (mean±standard deviation), UU IR mice had 3.5±2.9 seizures, and UU AC mice had 4.4±3.8 seizures during the 7-day monitoring period. The composition of the gut microbiome significantly differed in TMEV-infected mice fed the UU AC diet, with most measured differences occurring in Gram-positive bacteria. TMEV-infected mice fed the UU AC diet displayed worsened chronic working memory. Significance: Intestinal dysbiosis evokes stark differences in acute seizure presentation in the TMEV model and vastly influences the trajectory of post-TMEV infection-induced behavioral comorbidities of epilepsy. Our study reveals a novel disease-modifying contribution of intestinal bacterial species after TMEV-induced acute seizures.

Frontline Sodium Channel-Blocking Antiseizure Medicine Use Promotes Future Onset of Drug-Resistant Chronic Seizures.

The mechanisms of treatment-resistant epilepsy remain unclear. We have previously shown that frontline administration of therapeutic doses of lamotrigine (LTG), which preferentially inhibits the fast-inactivation state of sodium channels, during corneal kindling of mice promotes cross-resistance to several other antiseizure medicines (ASMs). However, whether this phenomenon extends to monotherapy with ASMs that stabilize the slow inactivation state of sodium channels is unknown. Therefore, this study assessed whether lacosamide (LCM) monotherapy during corneal kindling would promote future development of drug-resistant focal seizures in mice. Male CF-1 mice (n = 40/group; 18-25 g) were administered an anticonvulsant dose of LCM (4.5 mg/kg, i.p.), LTG (8.5 mg/kg, i.p.), or vehicle (0.5% methylcellulose) twice daily for two weeks during kindling. A subset of mice (n = 10/group) were euthanized one day after kindling for immunohistochemical assessment of astrogliosis, neurogenesis, and neuropathology. The dose-related antiseizure efficacy of distinct ASMs, including LTG, LCM, carbamazepine, levetiracetam, gabapentin, perampanel, valproic acid, phenobarbital, and topiramate, was then assessed in the remaining kindled mice. Neither LCM nor LTG administration prevented kindling: 29/39 vehicle-exposed mice were kindled; 33/40 LTG-exposed mice were kindled; and 31/40 LCM-exposed mice were kindled. Mice administered LCM or LTG during kindling became resistant to escalating doses of LCM, LTG, and carbamazepine. Perampanel, valproic acid, and phenobarbital were less potent in LTG- and LCM-kindled mice, whereas levetiracetam and gabapentin retained equivalent potency across groups. Notable differences in reactive gliosis and neurogenesis were also appreciated. This study indicates that early, repeated administration of sodium channel-blocking ASMs, regardless of inactivation state preference, promotes pharmacoresistant chronic seizures. Inappropriate ASM monotherapy in newly diagnosed epilepsy may thus be one driver of future drug resistance, with resistance being highly ASM class specific.

Continuous seizure emergency evoked in mice with pharmacological, electrographic, and pathological features distinct from status epilepticus.

OBJECTIVES: Benzodiazepines are the standard of care for the management of sustained seizure emergencies, including status epilepticus (SE) and seizure clusters. Seizure clusters are a variably defined seizure emergency wherein a patient has multiple seizures above a baseline rate, with intervening periods of recovery, distinguishing clusters from SE. Although these seizure emergencies are phenotypically distinct, the precise pathophysiological and mechanistic differences between SE and seizure clusters are understudied. Emergency-specific preclinical models may differentiate the behavioral and pathological mechanisms that are acutely associated with seizure emergencies and seizure termination to better manage these events. METHODS: Herein we characterize a novel model of sustained seizure emergency induced in CF-1 mice through the combined administration of high-dose phenytoin (PHT; 50 mg/kg, i.p.) and pentylenetetrazol (PTZ; 100 mg/kg, s.c.). RESULTS: We presently describe a mouse model of sustained seizure emergency that is pathologically, pharmacologically, and behaviorally distinct from SE. Acute administration of PHT 1 h prior to PTZ led to significantly more mice with unremitting continuous seizure activity (CSA; 73.4%) vs vehicle-pretreated mice (13.8%; p < .0001). CSA was sensitive to lorazepam and valproic acid when administered at seizure onset and 30 minutes later. Carbamazepine worsened seizure control and post-CSA survival. Mice in CSA exhibited electroencephalography (EEG) patterns distinct from kainic acid-induced SE and PTZ alone, clearly differentiating CSA from SE and PTZ-induced myoclonic seizures. Neuropathological assessment by Fluoro-Jade C staining of brains collected 24 h post-CSA revealed no neurodegeneration in any mouse that underwent CSA, whereas there was widespread neuronal death in brains from KA-SE mice. Finally, immunohistochemistry revealed acute seizure-induced astrogliosis (glial fibrillary acid protein; GFAP) in hippocampal structures, whereas hippocampal neuronal nuclei (NeuN) protein expression was only reduced in KA-SE mice. SIGNIFICANCE: We present a novel mouse model on which to further elucidate the mechanistic differences between sustained seizure emergencies (ie, SE and seizure clusters) to improve clinical interventions and define mechanisms of seizure termination.

A Subpopulation of Microglia Generated in the Adult Mouse Brain Originates from Prominin-1-Expressing Progenitors.

Microglia maintain brain health and play important roles in disease and injury. Despite the known ability of microglia to proliferate, the precise nature of the population or populations capable of generating new microglia in the adult brain remains controversial. We identified Prominin-1 (Prom1; also known as CD133) as a putative cell surface marker of committed brain myeloid progenitor cells. We demonstrate that Prom1-expressing cells isolated from mixed cortical cultures will generate new microglia in vitro To determine whether Prom1-expressing cells generate new microglia in vivo, we used tamoxifen inducible fate mapping in male and female mice. Induction of Cre recombinase activity at 10 weeks in Prom1-expressing cells leads to the expression of TdTomato in all Prom1-expressing progenitors and newly generated daughter cells. We observed a population of new TdTomato-expressing microglia at 6 months of age that increased in size at 9 months. When microglia proliferation was induced using a transient ischemia/reperfusion paradigm, little proliferation from the Prom1-expressing progenitors was observed with the majority of new microglia derived from Prom1-negative cells. Together, these findings reveal that Prom1-expressing myeloid progenitor cells contribute to the generation of new microglia both in vitro and in vivo Furthermore, these findings demonstrate the existence of an undifferentiated myeloid progenitor population in the adult mouse brain that expresses Prom1. We conclude that Prom1-expressing myeloid progenitors contribute to new microglia genesis in the uninjured brain but not in response to ischemia/reperfusion.SIGNIFICANCE STATEMENT Microglia, the innate immune cells of the CNS, can divide to slowly generate new microglia throughout life. Newly generated microglia may influence inflammatory responses to injury or neurodegeneration. However, the origins of the new microglia in the brain have been controversial. Our research demonstrates that some newly born microglia in a healthy brain are derived from cells that express the stem cell marker Prominin-1. This is the first time Prominin-1 cells are shown to generate microglia.

Development of an antiepileptogenesis drug screening platform: Effects of everolimus and phenobarbital.

OBJECTIVE: The kainic acid (KA)-induced status epilepticus (SE) model in rats is a well-defined model of epileptogenesis. This model closely recapitulates many of the clinical and pathological characteristics of human temporal lobe epilepsy (TLE) that arise following SE or another neurological insult. Spontaneous recurrent seizures (SRS) in TLE can present after a latent period following a neurological insult (traumatic brain injury, SE event, viral infection, etc.). Moreover, this model is suitable for preclinical studies to evaluate the long-term process of epileptogenesis and screen putative disease-modifying/antiepileptogenic agents. The burden of human TLE is highly variable, similar to the post-KA SE rat model. In this regard, this model may have broad translational relevance. This report thus details the pharmacological characterization and methodological refinement of a moderate-throughput drug screening program using the post-KA-induced SE model of epileptogenesis in male Sprague Dawley rats to identify potential agents that may prevent or modify the burden of SRS. Specifically, we sought to demonstrate whether our protocol could prevent the development of SRS or lead to a reduced frequency/severity of SRS. METHODS: Rats were administered either everolimus (2-3 mg/kg po) beginning 1, 2, or 24 h after SE onset, or phenobarbital (60 mg/kg ip) beginning 1 h after SE onset. All treatments were administered once/day for 5-7 days. Rats in all studies (n = 12/treatment dose/study) were then monitored intermittently by video-electroencephalography (2 weeks on, 2 weeks off, 2 weeks on epochs) to determine latency to onset of SRS and disease burden. RESULTS: Although no adverse side effects were observed in our studies, no treatment significantly modified disease or prevented the presentation of SRS by 6 weeks after SE onset. SIGNIFICANCE: Neither phenobarbital nor everolimus administered at several time points after SE onset prevented the development of SRS. Nonetheless, we demonstrate a practical and moderate-throughput screen for potential antiepileptogenic agents in a rat model of TLE.

Diurnal burden of spontaneous seizures in early epileptogenesis in the post-kainic acid rat model of epilepsy.

Patients with epilepsy can experience diurnal seizure patterns. However, few studies in rodent models of temporal lobe epilepsy (TLE) routinely quantify the diurnal pattern of spontaneous recurrent seizures (SRS), and those that have conducted such assessments used small groups. This study thus aimed to define whether there was a diurnal pattern of SRS in the early phases of epileptogenesis in a large cohort (n = 40) of post-kainic acid (KA)-induced status epilepticus (SE) male Sprague Dawley rats. Rats were monitored by continuous 24/7 video-EEG in two-week epochs up to 6 weeks post-KA-induced SE. The total number of SRS by 6 weeks post-SE correlated to body weight at the time of SE insult (R2  = .1465, P = .0143). The total number of spontaneous behavioral and electrographic seizures, seizure severity, and seizure burden was recorded during lights ON (light) or lights OFF (dark) phases. All measures significantly increased with time post-SE; we detected significantly more seizures during the lights OFF phase of the post-SE monitoring periods. Moreover, a subset of rats demonstrated marked seizure preference in the lights OFF phase. Our study confirms that a diurnal pattern of SRS is variably detectable in early epileptogenesis in this model of TLE.

Antiseizure drug efficacy and tolerability in established and novel drug discovery seizure models in outbred vs inbred mice.

OBJECTIVE: Initial identification of new investigational drugs for the treatment of epilepsy is commonly conducted in well-established mouse acute and chronic seizure models: for example, maximal electroshock (MES), 6 Hz, and corneal kindling. Comparison of the median effective dose (ED50) of approved antiseizure drugs (ASDs) vs investigational agents in these models provides evidence of their potential for clinical efficacy. Inbred and outbred mouse strains exhibit differential seizure susceptibility. However, few comparisons exist of the ED50 or median behaviorally impairing dose (TD50) of prototype ASDs in these models in inbred C57Bl/6 vs outbred CF-1 mice, both of which are often used for ASD discovery. METHODS: We defined the strain-related ED50s and TD50s of several mechanistically distinct ASDs across established acute seizure models (MES, 6 Hz, and corneal-kindled mouse). We further quantified the strain-related effect of the MES ED50 of each ASD on gross behavior in a locomotor activity assay. Finally, we describe a novel pharmacoresistant corneal-kindling protocol that is suitable for moderate-throughput ASD screening and demonstrates highly differentiated ASD sensitivity. RESULTS: We report significant strain-related differences in the MES ED50 of valproic acid (CF-1 ED50: 90 mg/kg [95% confidence interval (CI) 165-214] vs C57Bl/6: 276 mg/kg [226-366]), as well as significant differences in the ED50 of levetiracetam in the pharmacoresistant 6 Hz test (CF-1: 22.5 mg/kg [14.7-30.2] vs C57Bl/6: >500 mg/kg [CI not defined]). There were no differences in the calculated TD50 of these ASDs between strains. Furthermore, the MES ED50 of phenobarbital significantly enhanced locomotor activity of outbred CF-1, but not C57Bl/6, mice. SIGNIFICANCE: Altogether, this study provides strain-related information to differentiate investigational agents from ASD standards-of-care in commonly employed preclinical discovery models and describes a novel kindled seizure model to further explore the mechanisms of drug-resistant epilepsy.

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.

Cortisol is More Important than Metanephrines in Driving Changes in Leukocyte Counts after Stroke.

BACKGROUND: There are notable changes in the number of white blood cells (WBCs) after stroke, but the primary mediators of these changes are unclear. In this study, we assessed the role of the neuroendocrine and sympathetic nervous systems in stroke-induced changes of WBCs within distinct leukocyte subsets, as well as the effect of these changes on stroke outcomes. METHODS: Patients were recruited within 72 hours after ischemic stroke; complete blood count with differential was obtained at set time points. The relationships among leukocyte numbers, cortisol, adrenocorticotropic hormone, interleukin-6, and metanephrines were assessed at 72 hours after stroke. Associations between abnormal leukocyte counts at 72 hours, poststroke infection, and 3-month outcomes were determined. RESULTS: A total of 114 subjects were enrolled. Severe stroke was associated with leukocytosis, neutrophilia, monocytosis, lymphopenia, and eosinopenia. At 72 hours after stroke, increased serum cortisol was independently associated with neutrophilia and lymphopenia. Abnormal leukocyte counts were not independently predictive of poststroke infection, but lymphopenia was associated with poor outcome (modified Rankin score >3) at 3 months after stroke (odds ratio = 22.86 [1.95, 267.65]; P = .01). CONCLUSIONS: Increased serum cortisol is independently associated with neutrophilia and lymphopenia after stroke. Lymphopenia is not an independent predictor of infections but is independently associated with worse outcome.

Splenectomy Does Not Improve Long-Term Outcome After Stroke.

BACKGROUND AND PURPOSE: Immune responses to brain antigens after stroke contribute to poor outcome. We hypothesized that splenectomy would lessen the development of such responses and improve outcome. METHODS: Male Lewis rats (275-350 g) underwent 2-hour middle cerebral artery occlusion immediately after splenectomy or sham splenectomy. Animals were survived to 4 weeks (672 hrs), and immune responses to myelin basic protein determined at euthanasia. Infarct volume was determined in a subset of animals euthanized at 72 hours. Behavioral outcomes were assessed to 672 hours. RESULTS: Splenectomy was associated with worse neurological scores early after stroke, but infarct size at 72 hours was similar in both groups. Behavioral outcomes and immune responses to myelin basic protein were also similar among splenectomized and sham-operated animals 672 hours after middle cerebral artery occlusion. CONCLUSIONS: Splenectomy did not alter the immune responses to brain antigens or improve outcome after stroke. Differences between this study and other studies of splenectomy and stroke are examined.

Antibodies to myelin basic protein are associated with cognitive decline after stroke.

B lymphocytes cause post-stroke cognitive decline in mice. We therefore evaluated the association between autoantibodies and post-stroke cognitive decline in a prospectively collected human cohort. The mini-mental state exam (MMSE) was administered 30, 90, 180, and 365days after stroke. Antibody titers to myelin basic protein (MBP), proteolipid protein, and several non-specific proteins were determined. Among 58 subjects with initial MMSE≥20 and at least 2 MMSE examinations in the year after stroke, cognitive decline (MMSE decrease ≥2) occurred in 10 (17%) subjects. In multivariate analysis, MBP antibody titers were the only independent predictor of cognitive decline (OR=9.02 [1.18, 68.90]; P=0.03).

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.

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 ß-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.

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.

Poststroke fatigue: hints to a biological mechanism.

BACKGROUND: Poststroke fatigue (PSF) is common, but the biological basis of this fatigue is unknown. We explored the possibility that PSF is related to systemic inflammation by investigating polymorphisms in 2 genes that affect the immune response. METHODS: In a substudy of a larger trial that evaluated the role of the immune response on stroke outcome, fatigue was assessed at 30, 90, 180, and 365 days after ischemic stroke using the Fatigue Assessment Scale. Subjects were genotyped for 3 single nucleotide polymorphisms, one in the interleukin-1 receptor antagonist gene (IL1RN; rs4251961, a T/C substitution) and two in the in toll-like receptor-4 (TLR4) gene (1063 A/G [Asp299Gly] rs4986790 and 1363 C/T [Thr399Ile] rs4986791). RESULTS: Of the 39 participants, 22 (56%) endorsed fatigue during the study. The degree of fatigue was remarkably constant over time and independent of stroke outcome. The C allele of the rs4251961 single nucleotide polymorphism (SNP) in IL1RN was associated with self-reported fatigue (P = .03), whereas the cosegregating polymorphisms in TLR4 were associated with lower levels of fatigue (P= .04). CONCLUSIONS: SNPs in 2 genes with opposing effects on inflammatory immune responses were significantly, but differentially, associated with PSF. These findings suggest a direct link between immune signaling dysregulation and PSF.

Strain differences in fatigue and depression after experimental stroke.

Fatigue and depression are common symptoms after stroke. Animal models of poststroke fatigue (PSF) and poststroke depression (PSD) would facilitate the study of these symptoms. Spontaneous locomotor activity is as an objective measure of fatigue and learned helplessness an accepted correlate of depression. We used different rat strains to evaluate stroke-induced changes in behavior in hopes that interstrain differences would provide insights into the biological basis of these symptoms. Male Lewis, Wistar, and Sprague-Dawley (SD) rats underwent experimental stroke. Spontaneous activity was assessed continually after stroke (for up to 50 days). In a subset of animals, the forced swim test was performed prior to and 1 month after stroke to assess learned helplessness; blood was obtained at sacrifice for cytokine assay. Stroke induced strain-related differences in activity; Lewis rats increased spontaneous activity during the dark cycle, while Wistar and SD rats increased activity during the light cycle. The velocity of movement decreased during the dark cycle in Wistar and SD rats and during the light cycle in Lewis rats. Stroke also led to an increase in learned helplessness in Lewis rats. In summary, different patterns of behaviors emerge in different rat strains after stroke. Lewis rats displayed behavior consistent with depression but not fatigue, while Wistar and SD rats displayed behavior consistent with fatigue but not depression. These data argue that PSF and PSD are different biological constructs and suggest that analysis of strain-related differences may provide insight into symptom pathophysiology.

Functional polymorphisms in toll-like receptor 4 are associated with worse outcome in acute ischemic stroke patients.

Toll-like receptor-4 (TLR4) is important in neuroinflammation. Single nucleotide polymorphisms (SNPs) in TLR4, including 1063 A/G [Asp299Gly] and 1363 C/T [Thr399Ile], are associated with altered immune responses but their effect on acute ischemic stroke (AIS) outcome is unknown. We collected demographic, clinical, laboratory, radiologic, and genotype data on 113 AIS patients and performed multivariate analyses to assess associations between TLR4 SNP haplotype and either neurological outcome, infection, or inflammatory markers. In adjusted analyses, TLR4 SNPs were associated with worse outcome as well as increases in circulating leukocytes, C-reactive protein, and interleukin-1 receptor antagonist. In AIS, variations in TLR4 may influence neurological outcome (for video abstract, please see Supplemental digital content 1 file, http://links.lww.com/WNR/A274).

Variation in behavioral deficits and patterns of recovery after stroke among different rat strains.

Pre-clinical models of stroke therapeutics depend upon the ability to detect differences in infarct volume as well as in the short- and long-term outcomes of treated animals. Little attention has been paid to interstrain differences in these outcomes and the importance of defining the most appropriate behavioral tests. In this study, we evaluate long-term outcome from stroke in three different rat strains. Lewis, Wistar, and Sprague Dawley (SD) rats were subjected to 2-h middle cerebral artery occlusion and survived for up to 49 days. Behavioral tests were performed weekly. There was continuous assessment of rotational/circling activity in the home cage by use of an automated software program. A separate group of animals was sacrificed at 24 h to determine infarct volume. Infarct volume was similar in all three strains. Mortality was significantly higher in SD rats (P < 0.001). Rotational/circling activity at 24 h was correlated with cortical infarct volume in Wistar and SD rats (ρ = 0.67, P = 0.04 and ρ = 0.72, P = 0.01, respectively). Wistar and SD rats displayed more rotational/circling activity following stroke than Lewis rats, but Lewis rats evidenced more impairment on complex motor tasks like the rotarod. Further, computer automated analysis of rotational activity was more sensitive than subjective assessment, with SD rats showing a preference for clockwise rotations to 49 days after stroke despite normalization of the neurological score after 21 days. There are significant interstrain differences in survival and in the patterns of neurological impairment and recovery after stroke. These differences must be taken into account in pre-clinical studies, but may also be capitalized upon to understand genetic contributions to injury. Finally, computerized assessment of behavior is more sensitive than subjective assessment for detecting behavioral changes.

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.