Jessica B. Buchanan

Neuroinflammation and disruption in working memory in aged mice after acute stimulation of the peripheral innate immune system

Acute cognitive disorders are common in elderly patients with peripheral infections but it is not clear why. Here, we injected old and young mice with Escherichia coli lipopolysaccharide (LPS) to mimic an acute peripheral infection and separated the hippocampal neuronal cell layers from the surrounding hippocampal tissue by laser capture microdissection and measured mRNA for several inflammatory cytokines (IL-1 beta, IL-6, and TNFalpha) that are known to disrupt cognition. The results showed that old mice had an increased inflammatory response in the hippocampus after LPS compared to younger cohorts. Immunohistochemistry further showed more microglial cells in the hippocampus of old mice compared to young adults, and that more IL-1 beta-positive cells were present in the dentate gyrus and in the CA1, CA2, and CA3 regions of LPS-treated old mice compared to young adults. In a test of cognition that required animals to effectively integrate new information with a preexisting schema to complete a spatial task, we found that hippocampal processing is more easily disrupted in old animals than in younger ones when the peripheral innate immune system is stimulated. Collectively, the results suggest that aging can facilitate neurobehavioral complications associated with peripheral infections probably by allowing the over expression of inflammatory cytokines in brain areas that mediate cognitive processing.

Interleukin-6 Facilitates Lipopolysaccharide-Induced Disruption in Working Memory and Expression of Other Proinflammatory Cytokines in Hippocampal Neuronal Cell Layers

Proinflammatory cytokines inhibit learning and memory but the significance of interleukin-6 (IL-6) in acute cognitive deficits induced by the peripheral innate immune system is not known. To examine the functional role of IL-6 in hippocampus-mediated cognitive impairments associated with peripheral infections, C57BL6/J (IL-6+/+) and IL-6 knock-out (IL-6−/−) mice were trained in a matching-to-place version of the water maze. After an acquisition phase, IL-6+/+ mice injected intraperitoneally with lipopolysaccharide (LPS) exhibited deficits in working memory. However, IL-6−/− mice were refractory to the LPS-induced impairment in working memory. To determine the mechanism by which IL-6 deficiency conferred protection from disruption in working memory, plasma IL-1β and tumor necrosis factor α (TNFα), c-Fos immunoreactivity in the nucleus of the solitary tract (NTS), and steady-state levels of IL-1β and TNFα mRNA in neuronal layers of the hippocampus were determined in IL-6+/+ and IL-6−/− mice after injection of LPS. Plasma IL-1β and TNFα and c-Fos immunoreactivity in the NTS were increased similarly in IL-6+/+ and IL-6−/− mice after LPS, indicating high circulating levels of IL-1β and TNFα and activation of vagal afferent pathways were not sufficient to disrupt working memory in the absence of IL-6. However, the LPS-induced upregulation of IL-1β and TNFα mRNA that was evident in hippocampal tissue of IL-6+/+ mice was greatly attenuated or entirely absent in IL-6−/− mice. Collectively, these data suggest that humoral and neural immune-to-brain communication pathways are intact in IL-6-deficient mice but that, in the absence of IL-6, the central cytokine compartment is hyporesponsive.

Cognitive and neuroinflammatory consequences of mild repeated stress are exacerbated in aged mice.

Peripheral immune stimulation as well as certain types of psychological stress increases brain levels of inflammatory cytokines such as interleukin-1beta (IL-1beta), IL-6 and tumor necrosis factor alpha (TNFalpha). We have demonstrated that aged mice show greater increases in central inflammatory cytokines, as well as greater cognitive deficits, compared to adults in response to peripheral lipopolysaccharide (LPS) administration. Because aged mice are typically more sensitive to systemic stressors such as LPS, and certain psychological stressors induce physiological responses similar to those that follow LPS, we hypothesized that aged mice would be more sensitive to the physiological and cognitive effects of mild stress than adult mice. Here, adult (3-5 months) and aged (22-23 months) male BALB/c mice were trained in the Morris water maze for 5 days. Mice were then exposed to a mild restraint stress of 30 min before being tested in a working memory version of the water maze over a 3-day period. On day 4 mice were stressed and then killed for collection of blood and brain. In a separate group of animals, mice were killed immediately after one, two or three 30 min restraint sessions and blood was collected for peripheral corticosterone and cytokine protein measurement, and brains were dissected for central cytokine mRNA measurement. Stress disrupted spatial working memory in both adult and aged mice but to a much greater extent in the aged mice. In addition, aged mice showed an increase in stress-induced expression of hippocampal IL-1beta mRNA and MHC class II protein compared to non-stressed controls while expression in adult mice was unaffected by stress. These data show that aged mice are more sensitive to both the cognitive and inflammatory effects of mild stress than are adult mice and suggest a possible role for IL-1beta.

Cognitive deficits in interleukin-10-deficient mice after peripheral injection of lipopolysaccharide

Interleukin (IL)-10 is important for regulating inflammation but whether it protects against infection-related deficits in cognitive function is unknown. Therefore, the current study evaluated sickness behavior, hippocampal-dependent matching-to-place performance and several inflammatory cytokines and neurotrophins in wild-type (IL-10(+/+)) and IL-10-deficient (IL-10(-/-)) mice after i.p. injection of lipopolysaccharide (LPS). Additionally, morphology of dendrites of pyramidal neurons in the dorsal CA1 hippocampus was assessed. Treatment with LPS increased IL-1beta, IL-6, and tumor necrosis factor alpha (TNFalpha) mRNA in all brain areas examined including the hippocampus, in both IL-10(+/+) and IL-10(-/-) mice but the increase was largest in IL-10(-/-) mice. Plasma IL-1beta, IL-6 and TNFalpha were also higher in IL-10(-/-) mice compared to IL-10(+/+) mice after LPS. Consistent with increased inflammatory cytokines in IL-10(-/-) mice after LPS treatment, were a more lengthy sickness behavior syndrome and a more prominent reduction in hippocampal levels of nerve growth factor mRNA; brain-derived neurotrophic factor mRNA was reduced similarly in both genotypes after LPS. In a test of hippocampal-dependent learning and memory that required mice to integrate new information with previously learned information and switch strategies to master a task, IL-10(-/-) mice were found to be less efficient after LPS than were similarly treated wild-type mice. LPS did not affect morphology of dendrites of pyramidal neurons in the dorsal CA1 hippocampus in either genotype. Taken together the results are interpreted to suggest that during peripheral infection IL-10 inhibits sickness behavior and tribulations in hippocampal-dependent working memory via its propensity to mitigate inflammation. We conclude that IL-10 is critical for maintaining normal neuro-immune communication during infection.

Architectural changes to CA1 pyramidal neurons in adult and aged mice after peripheral immune stimulation

The expression of several inflammatory cytokines that inhibit synaptic plasticity and hippocampal-dependent learning and memory is higher in the brains of aged mice compared to young adults after peripheral injection of lipopolysaccharide (LPS). In this study we investigated whether the exaggerated inflammatory cytokine response in the hippocampus of aged mice after IP injection of LPS is associated with architectural changes to dendrites of pyramidal neurons in the dorsal CA1 hippocampus. Compared to young adults, aged mice had higher basal expression of MHC class II, lower basal expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and a decrease in total dendritic length in both the basal and apical tree. After IP LPS administration, expression of IL-1beta, IL-6, and TNFalpha mRNA was higher in hippocampus of aged mice compared to young adults whereas NGF and BDNF mRNA was reduced similarly in both age groups. The basal dendritic tree was not affected by LPS in either adult or aged mice 72h after treatment; however, length and branching of the apical tree was reduced by LPS in aged but not adult mice. The present findings indicate that a peripheral infection in the aged can cause a heightened inflammatory cytokine response in the hippocampus and atrophy of hippocampal neurons. Architectural changes to dorsal CA1 hippocampal neurons may contribute to cognitive disorders evident in elderly patients with an infection.

Exacerbated fatigue and motor deficits in interleukin-10-deficient mice after peripheral immune stimulation

The anti-inflammatory cytokine interleukin (IL)-10 is important for regulating inflammation in the periphery and brain, but whether it protects against infection- or age-related psychomotor disturbances and fatigue is unknown. Therefore, the present study evaluated motor coordination, time to fatigue, and several central and peripheral proinflammatory cytokines in male young adult (3-mo-old) and middle-aged (12-mo-old) wild-type (IL-10(+/+)) and IL-10-deficient (IL-10(-/-)) mice after intraperitoneal injection of lipopolysaccharide (LPS) or saline. No age-related differences were observed; therefore, data from the two ages were pooled and analyzed to determine effects of genotype and treatment. LPS treatment increased IL-1beta, IL-6, and TNFalpha mRNA in all brain areas examined in IL-10(+/+) and IL-10(-/-) mice, but to a greater extent and for a longer time in IL-10(-/-) mice. Plasma IL-1beta and IL-6 were increased similarly in IL-10(+/+) and IL-10(-/-) mice 4 h after LPS but remained elevated longer in IL-10(-/-) mice, whereas TNFalpha was higher in IL-10(-/-) mice throughout after LPS treatment. Motor performance and motor learning in IL-10(+/+) mice were not affected by LPS treatment; however, both were reduced in IL-10(-/-) mice treated with LPS compared with those treated with saline. Furthermore, although LPS reduced the time to fatigue in IL-10(+/+) and IL-10(-/-) mice, the effects were exacerbated in IL-10(-/-) mice. Thus the increased brain and peripheral inflammation induced by LPS in IL-10(-/-) mice was associated with increased coordination deficits and fatigue. These data suggest that IL-10 may inhibit motor deficits and fatigue associated with peripheral infections via its anti-inflammatory effects.

Regulation of Food Intake by Inflammatory Cytokines in the Brain

A number of inflammatory cytokines are synthesized and released after activation of the immune system. In addition to other biological effects, these cytokines can potently inhibit food intake. Cytokine-mediated inhibition of food intake is of particular importance because excessive production of peripheral inflammatory cytokines is often associated with the cachexia-anorexia syndrome seen in some chronic diseases. The weight loss in cachexia is associated with an increase in morbidity and mortality. Understanding how cytokines regulate food intake may be crucial in enhancing quality of life and facilitating recovery in patients exhibiting cachexia. This review describes the main inflammatory cytokines that influence food intake and explores how peripheral cytokines communicate with hypothalamic nuclei to influence feeding.

Methamphetamine sensitization attenuates the febrile and neuroinflammatory response to a subsequent peripheral immune stimulus.

Methamphetamine (MA) use is associated with activation of microglia and, at high doses, can induce neurotoxicity. Given the changes in the neuroinflammatory environment associated with MA, we investigated whether MA sensitization, a model of stimulant psychosis and an indicator of drug addiction, would interfere with the thermoregulatory and neuroinflammatory response to a subsequent peripheral immune stimulus. C57BL6/J mice were given either 1 mg/kg MA or saline i.p. once a day for 5 days to produce behavioral sensitization. Seventy-two hours following the last MA injection, 100 microg/kg LPS or saline was co-administered with 1 mg/kg MA or saline and blood and brains were collected. Here we report that while co-administration of LPS and MA did not affect the LPS-induced increase in central cytokine mRNA, mice sensitized to MA showed an attenuated central response to LPS. Interestingly, the peripheral response to LPS was not affected by MA sensitization. Plasma cytokines increased similarly in all groups after LPS. Further, c-Fos expression in the nucleus of the solitary tract did not differ between groups, suggesting that the periphery-to-brain immune signal is intact in MA-sensitized mice and that the deficit lies in the central cytokine compartment. We also show that MA sensitization decreased LPS- or acute MA-induced microglial Iba1 expression compared to non-sensitized mice. Taken together, these data show that MA sensitization interferes with the normal central immune response, preventing the CNS from efficiently responding to signals from the peripheral immune system.

A neurotoxic regimen of methamphetamine exacerbates the febrile and neuroinflammatory response to a subsequent peripheral immune stimulus

Methamphetamine (MA) use is associated with activation of microglia and, at high doses, can induce neurotoxicity. Given the changes in the neuroinflammatory environment associated with MA, we investigated whether MA administration would interfere with the thermoregulatory and neuroinflammatory response to a subsequent peripheral immune stimulus. C57BL6/J mice were given four i.p. injections of either 5 mg/kg MA or saline at two hour intervals. Twenty-four hours following the first MA injection, mice were given 100 μg/kg LPS or saline i.p. and blood and brains were collected. Here we report that mice exposed to MA developed higher fevers in response to LPS than did those given LPS alone. MA also exacerbated the LPS-induced increase in central cytokine mRNA. MA alone increased microglial Iba1 expression and expression was further increased when mice were exposed to both MA and LPS, suggesting that MA not only activated microglia but also influenced their response to a peripheral immune stimulus. Taken together, these data show that MA administration exacerbates the normal central immune response, most likely by altering microglia.

A warmer ambient temperature increases the passage of interleukin-1β into the brains of old rats

We have demonstrated that after intraperitoneal lipopolysaccharide (LPS) injection, old rats mount fevers similar to those of young rats at an ambient temperature (Ta) of 31 degrees C, but not at 21 degrees C. The same is true for intraperitoneal or intravenous IL-1beta administration. The underlying mechanism responsible for blunted fever in old rats may be a deficiency in communication between the periphery and the brain. Possibly, peripheral cytokine actions are altered in old rats, such that the signal that reaches the brain is diminished. Here, we hypothesized that at standard laboratory temperatures, not enough IL-1beta is reaching the brain for fever to occur and that a warmer Ta would increase the influx of IL-1beta into the brain, enabling old rats to generate fever. Young (3-5 mo) and old (23-29 mo) Long-Evans rats were maintained for 3 days at either Ta 21 or 31 degrees C prior to intravenous injection with radiolabeled IL-1beta to measure passage across the blood-brain barrier. Young rats showed similar influx of IL-1beta into the brain at the two Tas, but old rats showed significant influx only at the warmer Ta. These data suggest that the lack of fever at a cool Ta may be due to a reduced influx of IL-1beta into the brain.