Early life cancer and chemotherapy lead to cognitive deficits related to alterations in microglial-associated gene expression in prefrontal cortex.

Children that survive leukemia are at an increased risk for cognitive difficulties. A better understanding of the neurobiological changes in response to early life chemotherapy will help develop therapeutic strategies to improve quality of life for leukemia survivors. To that end, we used a translationally-relevant mouse model consisting of leukemic cell line (L1210) injection into postnatal day (P)19 mice followed by methotrexate, vincristine, and leucovorin chemotherapy. Beginning one week after the end of chemotherapy, social behavior, recognition memory and executive function (using the 5 choice serial reaction time task (5CSRTT)) were tested in male and female mice. Prefrontal cortex (PFC) and hippocampus (HPC) were collected at the conclusion of behavioral assays for gene expression analysis. Mice exposed to early life cancer + chemotherapy were slower to progress through increasingly difficult stages of the 5CSRTT and showed an increase in premature errors, indicating impulsive action. A cluster of microglial-related genes in the PFC were found to be associated with performance in the 5CSRTT and acquisition of the operant response, and long-term changes in gene expression were evident in both PFC and HPC. This work identifies gene expression changes in PFC and HPC that may underlie cognitive deficits in survivors of early life exposure to cancer + chemotherapy.

Perinatal Morphine Exposure Leads to Sex-Dependent Executive Function Deficits and Microglial Changes in Mice.

Children exposed prenatally to opioids are at an increased risk for behavioral problems and executive function deficits. The prefrontal cortex (PFC) and amygdala (AMG) regulate executive function and social behavior and are sensitive to opioids prenatally. Opioids can bind to toll-like receptor 4 (TLR4) to activate microglia, which may be developmentally important for synaptic pruning. Therefore, we tested the effects of perinatal morphine exposure on executive function and social behavior in male and female mouse offspring, along with microglial-related and synaptic-related outcomes. Dams were injected once daily subcutaneously with saline (n = 8) or morphine (MO; 10 mg/kg; n = 12) throughout pregestation, gestation, and lactation until offspring were weaned on postnatal day 21 (P21). Male MO offspring had impairments in attention and accuracy in the five-choice serial reaction time task, while female MO offspring were less affected. Targeted gene expression analysis at P21 in the PFC identified alterations in microglial-related and TLR4-related genes, while immunohistochemical analysis in adult brains indicated decreased microglial Iba1 and phagocytic CD68 proteins in the PFC and AMG in males, but females had an increase. Further, both male and female MO offspring had increased social preference. Overall, these data demonstrate male vulnerability to executive function deficits in response to perinatal opioid exposure and evidence for disruptions in neuron-microglial signaling.

CXCL13 expressed on inflamed cerebral blood vessels recruit IL-21 producing TFH cells to damage neurons following stroke.

BACKGROUND: Ischemic stroke is a leading cause of mortality worldwide, largely due to the inflammatory response to brain ischemia during post-stroke reperfusion. Despite ongoing intensive research, there have not been any clinically approved drugs targeting the inflammatory component to stroke. Preclinical studies have identified T cells as pro-inflammatory mediators of ischemic brain damage, yet mechanisms that regulate the infiltration and phenotype of these cells are lacking. Further understanding of how T cells migrate to the ischemic brain and facilitate neuronal death during brain ischemia can reveal novel targets for post-stroke intervention. METHODS: To identify the population of T cells that produce IL-21 and contribute to stroke, we performed transient middle cerebral artery occlusion (tMCAO) in mice and performed flow cytometry on brain tissue. We also utilized immunohistochemistry in both mouse and human brain sections to identify cell types and inflammatory mediators related to stroke-induced IL-21 signaling. To mechanistically demonstrate our findings, we employed pharmacological inhibitor anti-CXCL13 and performed histological analyses to evaluate its effects on brain infarct damage. Finally, to evaluate cellular mechanisms of stroke, we exposed mouse primary neurons to oxygen glucose deprivation (OGD) conditions with or without IL-21 and measured cell viability, caspase activity and JAK/STAT signaling. RESULTS: Flow cytometry on brains from mice following tMCAO identified a novel population of cells IL-21 producing CXCR5+ CD4+ ICOS-1+ T follicular helper cells (TFH) in the ischemic brain early after injury. We observed augmented expression of CXCL13 on inflamed brain vascular cells and demonstrated that inhibition of CXCL13 protects mice from tMCAO by restricting the migration and influence of IL-21 producing TFH cells in the ischemic brain. We also illustrate that neurons express IL-21R in the peri-infarct regions of both mice and human stroke tissue in vivo. Lastly, we found that IL-21 acts on mouse primary ischemic neurons to activate the JAK/STAT pathway and induce caspase 3/7-mediated apoptosis in vitro. CONCLUSION: These findings identify a novel mechanism for how pro-inflammatory T cells are recruited to the ischemic brain to propagate stroke damage and provide a potential new therapeutic target for stroke.

Translationally relevant mouse model of early life cancer and chemotherapy exposure results in brain and small intestine cytokine responses: A potential link to cognitive deficits.

Survivors of acute lymphoblastic leukemia (ALL), the most common childhood cancer, are at increased risk for long-term cognitive problems, including executive function deficits. The chemotherapeutic agent methotrexate (MTX) is used to treat most ALL patients and is closely associated with cognitive deficits. To address how early life cancer chemotherapy leads to cognitive deficits, we developed a translationally relevant mouse model of leukemia survival that exposed mice to leukemic cells and chemotherapeutic drugs (vincristine and MTX, with leucovorin rescue) in early life. Male and female mice were tested several weeks later using novel object recognition (recognition memory) and 5-choice serial reaction time task (executive function). Gene expression of proinflammatory, white matter and synapse-associated molecules was assessed in the prefrontal cortex and small intestine both acutely after chemotherapy and chronically after cognitive testing. Early life cancer-chemotherapy exposure resulted in recognition memory and executive function deficits in adult male mice. Prefrontal cortex expression of the chemokine Ccl2 was increased acutely, while small intestine expression of the proinflammatory cytokine tumor necrosis factor-alpha was elevated both acutely (both sexes) and chronically (males only). Inflammation in the small intestine was correlated with prefrontal cortical proinflammatory and synaptic gene expression changes, as well as to executive function deficits. Collectively, these data indicate that the current protocol results in a robust mouse model in which to study cognitive deficits in leukemia survivors, and suggest that small intestine inflammation may represent a novel contributor to adverse CNS consequences of early life chemotherapy.

Adolescent microglia play a role in executive function in male mice exposed to perinatal high fat diet.

In humans, excessive gestational weight gain during pregnancy is associated with an increased risk for executive function deficits in the offspring. Our previous work has confirmed this finding in mice, as offspring from dams fed a 60% high fat (HF) diet during breeding, gestation, and lactation demonstrate impulsive-like behavior in the 5 choice serial reaction time task (5CSRTT). Because the prefrontal cortex (PFC), which plays a key role in executive function, undergoes substantial postnatal adolescent pruning and microglia are actively involved in synaptic refinement, we hypothesized that microglia may play a role in mediating changes in brain development after maternal HF diet, with a specific focus on microglial activity during adolescence. Therefore, we treated male and female offspring from HF or control diet (CD) dams with PLX3397-formulated diet (PLX) to ablate microglia during postnatal days 23-45. After PLX removal and microglial repopulation, adult mice underwent testing to evaluate executive function. Adolescent PLX treatment did increase the control male dropout rate in learning the basic FR1 task, but otherwise had a minimal effect on behavior in control offspring. In males, HF offspring learned faster and performed better on a simple operant task (fixed ratio 1) without an effect of PLX. However, in HF offspring this increase in FR1 responding was associated with more impulsive errors in the 5CSRTT while PLX eliminated this association and decreased impulsive errors specifically in HF offspring. This suggests that adolescent PLX treatment improves executive function and particularly impulsive behavior in adult male HF offspring, without an overall effect of perinatal diet. In females, maternal HF diet impaired reversal learning but PLX had no effect on performance. We then measured gene expression in adult male PFC, nucleus accumbens (NAC), and amygdala (AMG), examining targets related to synaptic function, reward, and inflammation. Maternal HF diet increased PFC synaptophysin and AMG psd95 expression. PFC synaptophysin expression was correlated with more impulsive errors in the 5CSRTT in the HF offspring only and PLX treatment eliminated this correlation. These data suggest that adolescent microglia may play a critical role in mediating executive function after perinatal high fat diet in males.