Pregnancy Outcomes in Patients Exposed to OnabotulinumtoxinA Treatment: A Cumulative 29-Year Safety Update.

BACKGROUND AND OBJECTIVES: A previous publication of pregnancy outcomes in onabotulinumtoxinA-exposed mothers demonstrated that the prevalence of major fetal defects (0.9%, 1/110) was comparable with background rates in the general population. There is continued interest to better understand the safety of onabotulinumtoxinA during pregnancy. This analysis evaluated pregnancy outcomes after onabotulinumtoxinA exposure to provide a cumulative 29-year update. METHODS: The Allergan Global Safety Database was searched from January 1, 1990, to December 31, 2018. Data from women (younger than 65 years or unknown) during pregnancy or ≤3 months before conception treated with onabotulinumtoxinA were assessed to estimate birth defect prevalence rates of live births only from prospective pregnancies. RESULTS: Of 913 pregnancies, 397 (43.5%) were eligible with known outcomes. Maternal age was known in 215 pregnancies: 45.6% were 35 years or older. Indication was known in 340 pregnancies: most frequent were aesthetic (35.3%) and migraine/headache (30.3%). The timing of exposure was known in 318 pregnancies: 94.6% were before conception or during the first trimester. OnabotulinumtoxinA dose information was known in 242 pregnancies; most (83.5%) were exposed to <200 U. Of 195 prospective pregnancies with 197 fetuses, there were 152 (77.2%) live births and 45 (22.8%) fetal losses (32 spontaneous, 13 elective). Of 152 live births, 148 (97.4%) had normal outcomes and 4 had abnormal outcomes. Among the 4 abnormal outcomes, there were 1 major birth defect, 2 minor fetal defects, and 1 birth complication. The prevalence rate for overall fetal defects was 2.6% (4/152, 95% CI 1.0%-6.6%) and 0.7% (1/152, 95% CI 0.1%-3.6%) for major fetal defects (3%-6% in the general population). Among cases of live births and known determinable exposure times, there was 1 birth defect with preconception exposure and 2 with first-trimester exposure. DISCUSSION: Although subject to reporting bias due to the nature of the postmarketing database review, this 29-year retrospective analysis of safety data in pregnant women exposed to onabotulinumtoxinA demonstrates that the prevalence rate of major fetal defects among live births is consistent with the rates reported in the general population. Although there are limited data available for second-trimester and third-trimester exposure, this updated and expanded safety analysis provides important real-world evidence to health care providers and their patients. CLASSIFICATION OF EVIDENCE: This analysis provides Class III data that demonstrate that the prevalence rate of major fetal defects among live births subsequent to in utero onabotulinumtoxinA exposure is comparable with the reported background rates.

Microglial dyshomeostasis drives perineuronal net and synaptic loss in a CSF1R+/- mouse model of ALSP, which can be rescued via CSF1R inhibitors.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia is an autosomal dominant neurodegenerative disease caused by mutations in colony-stimulating factor 1 receptor (CSF1R). We sought to identify the role of microglial CSF1R haploinsufficiency in mediating pathogenesis. Using an inducible Cx3cr1 CreERT2/+-Csf1r +/fl system, we found that postdevelopmental, microglia-specific Csf1r haploinsufficiency resulted in reduced expression of homeostatic microglial markers. This was associated with loss of presynaptic surrogates and the extracellular matrix (ECM) structure perineuronal nets. Similar phenotypes were observed in constitutive global Csf1r haploinsufficient mice and could be reversed/prevented by microglia elimination in adulthood. As microglial elimination is unlikely to be clinically feasible for extended durations, we treated adult CSF1R+/- mice at different disease stages with a microglia-modulating dose of the CSF1R inhibitor PLX5622, which prevented microglial dyshomeostasis along with synaptic- and ECM-related deficits. These data highlight microglial dyshomeostasis as a driver of pathogenesis and show that CSF1R inhibition can mitigate these phenotypes.

Altered Hippocampal Epigenetic Regulation Underlying Reduced Cognitive Development in Response to Early Life Environmental Insults.

The hippocampus is involved in learning and memory and undergoes significant growth and maturation during the neonatal period. Environmental insults during this developmental timeframe can have lasting effects on brain structure and function. This study assessed hippocampal DNA methylation and gene transcription from two independent studies reporting reduced cognitive development stemming from early life environmental insults (iron deficiency and porcine reproductive and respiratory syndrome virus (PRRSv) infection) using porcine biomedical models. In total, 420 differentially expressed genes (DEGs) were identified between the reduced cognition and control groups, including genes involved in neurodevelopment and function. Gene ontology (GO) terms enriched for DEGs were associated with immune responses, angiogenesis, and cellular development. In addition, 116 differentially methylated regions (DMRs) were identified, which overlapped 125 genes. While no GO terms were enriched for genes overlapping DMRs, many of these genes are known to be involved in neurodevelopment and function, angiogenesis, and immunity. The observed altered methylation and expression of genes involved in neurological function suggest reduced cognition in response to early life environmental insults is due to altered cholinergic signaling and calcium regulation. Finally, two DMRs overlapped with two DEGs, VWF and LRRC32, which are associated with blood brain barrier permeability and regulatory T-cell activation, respectively. These results support the role of altered hippocampal DNA methylation and gene expression in early life environmentally-induced reductions in cognitive development across independent studies.

Replacement of microglia in the aged brain reverses cognitive, synaptic, and neuronal deficits in mice.

Microglia, the resident immune cell of the brain, can be eliminated via pharmacological inhibition of the colony-stimulating factor 1 receptor (CSF1R). Withdrawal of CSF1R inhibition then stimulates microglial repopulation, effectively replacing the microglial compartment. In the aged brain, microglia take on a "primed" phenotype and studies indicate that this coincides with age-related cognitive decline. Here, we investigated the effects of replacing the aged microglial compartment with new microglia using CSF1R inhibitor-induced microglial repopulation. With 28 days of repopulation, replacement of resident microglia in aged mice (24 months) improved spatial memory and restored physical microglial tissue characteristics (cell densities and morphologies) to those found in young adult animals (4 months). However, inflammation-related gene expression was not broadly altered with repopulation nor the response to immune challenges. Instead, microglial repopulation resulted in a reversal of age-related changes in neuronal gene expression, including expression of genes associated with actin cytoskeleton remodeling and synaptogenesis. Age-related changes in hippocampal neuronal complexity were reversed with both microglial elimination and repopulation, while microglial elimination increased both neurogenesis and dendritic spine densities. These changes were accompanied by a full rescue of age-induced deficits in long-term potentiation with microglial repopulation. Thus, several key aspects of the aged brain can be reversed by acute noninvasive replacement of microglia.

Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-ß pathology.

In addition to amyloid-ß plaque and tau neurofibrillary tangle deposition, neuroinflammation is considered a key feature of Alzheimer's disease pathology. Inflammation in Alzheimer's disease is characterized by the presence of reactive astrocytes and activated microglia surrounding amyloid plaques, implicating their role in disease pathogenesis. Microglia in the healthy adult mouse depend on colony-stimulating factor 1 receptor (CSF1R) signalling for survival, and pharmacological inhibition of this receptor results in rapid elimination of nearly all of the microglia in the central nervous system. In this study, we set out to determine if chronically activated microglia in the Alzheimer's disease brain are also dependent on CSF1R signalling, and if so, how these cells contribute to disease pathogenesis. Ten-month-old 5xfAD mice were treated with a selective CSF1R inhibitor for 1 month, resulting in the elimination of ∼80% of microglia. Chronic microglial elimination does not alter amyloid-ß levels or plaque load; however, it does rescue dendritic spine loss and prevent neuronal loss in 5xfAD mice, as well as reduce overall neuroinflammation. Importantly, behavioural testing revealed improvements in contextual memory. Collectively, these results demonstrate that microglia contribute to neuronal loss, as well as memory impairments in 5xfAD mice, but do not mediate or protect from amyloid pathology.

Colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xTg-AD mice.

BACKGROUND: Microglia are dependent upon colony-stimulating factor 1 receptor (CSF1R) signaling for their survival in the adult brain, with administration of the dual CSF1R/c-kit inhibitor PLX3397 leading to the near-complete elimination of all microglia brainwide. Here, we determined the dose-dependent effects of a specific CSF1R inhibitor (PLX5622) on microglia in both wild-type and the 3xTg-AD mouse model of Alzheimer's disease. METHODS: Wild-type mice were treated with PLX5622 for up to 21 days, and the effects on microglial numbers were assessed. 3xTg-AD mice were treated with PLX5622 for 6 or 12 weeks and effects on microglial numbers and pathology subsequently assessed. RESULTS: High doses of CSF1R inhibitor eliminate most microglia from the brain, but a 75% lower-dose results in sustained elimination of ~30 of microglia in both wild-type and 3xTg-AD mice. No behavioral or cognitive deficits were found in mice either depleted of microglia or treated with lower CSF1R inhibitor concentrations. Aged 3xTg-AD mice treated for 6 or 12 weeks with lower levels of PLX5622 resulted in improved learning and memory. Aß levels and plaque loads were not altered, but microglia in treated mice no longer associated with plaques, revealing a role for the CSF1R in the microglial reaction to plaques, as well as in mediating cognitive deficits. CONCLUSIONS: We find that inhibition of CSF1R alone is sufficient to eliminate microglia and that sustained microglial elimination is concentration-dependent. Inhibition of the CSF1R at lower levels in 3xTg-AD mice prevents microglial association with plaques and improves cognition.

Characterizing newly repopulated microglia in the adult mouse: impacts on animal behavior, cell morphology, and neuroinflammation.

Microglia are the primary immune cell in the brain and are postulated to play important roles outside of immunity. Administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor, PLX3397, to adult mice results in the elimination of ~99% of microglia, which remain eliminated for as long as treatment continues. Upon removal of the inhibitor, microglia rapidly repopulate the entire adult brain, stemming from a central nervous system (CNS) resident progenitor cell. Using this method of microglial elimination and repopulation, the role of microglia in both healthy and diseased states can be explored. Here, we examine the responsiveness of newly repopulated microglia to an inflammatory stimulus, as well as determine the impact of these cells on behavior, cognition, and neuroinflammation. Two month-old wild-type mice were placed on either control or PLX3397 diet for 21 d to eliminate microglia. PLX3397 diet was then removed in a subset of animals to allow microglia to repopulate and behavioral testing conducted beginning at 14 d repopulation. Finally, inflammatory profiling of the microglia-repopulated brain in response to lipopolysaccharide (LPS; 0.25 mg/kg) or phosphate buffered saline (PBS) was determined 21 d after inhibitor removal using quantitative real time polymerase chain reaction (RT-PCR), as well as detailed analyses of microglial morphologies. We find mice with repopulated microglia to perform similarly to controls by measures of behavior, cognition, and motor function. Compared to control/resident microglia, repopulated microglia had larger cell bodies and less complex branching in their processes, which resolved over time after inhibitor removal. Inflammatory profiling revealed that the mRNA gene expression of repopulated microglia was similar to normal resident microglia and that these new cells appear functional and responsive to LPS. Overall, these data demonstrate that newly repopulated microglia function similarly to the original resident microglia without any apparent adverse effects in healthy adult mice.

Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain.

The colony-stimulating factor 1 receptor (CSF1R) is a key regulator of myeloid lineage cells. Genetic loss of the CSF1R blocks the normal population of resident microglia in the brain that originates from the yolk sac during early development. However, the role of CSF1R signaling in microglial homeostasis in the adult brain is largely unknown. To this end, we tested the effects of selective CSF1R inhibitors on microglia in adult mice. Surprisingly, extensive treatment results in elimination of ∼99% of all microglia brain-wide, showing that microglia in the adult brain are physiologically dependent upon CSF1R signaling. Mice depleted of microglia show no behavioral or cognitive abnormalities, revealing that microglia are not necessary for these tasks. Finally, we discovered that the microglia-depleted brain completely repopulates with new microglia within 1 week of inhibitor cessation. Microglial repopulation throughout the CNS occurs through proliferation of nestin-positive cells that then differentiate into microglia.

Respiratory viral infection in neonatal piglets causes marked microglia activation in the hippocampus and deficits in spatial learning.

Environmental insults during sensitive periods can affect hippocampal development and function, but little is known about peripheral infection, especially in humans and other animals whose brain is gyrencephalic and experiences major perinatal growth. Using a piglet model, the present study showed that inoculation on postnatal day 7 with the porcine reproductive and respiratory syndrome virus (PRRSV) caused microglial activation within the hippocampus with 82% and 43% of isolated microglia being MHC II(+) 13 and 20 d after inoculation, respectively. In control piglets, <5% of microglia isolated from the hippocampus were MHC II(+). PRRSV piglets were febrile (p < 0.0001), anorectic (p < 0.0001), and weighed less at the end of the study (p = 0.002) compared with control piglets. Increased inflammatory gene expression (e.g., IL-1ß, IL-6, TNF-α, and IFN-γ) was seen across multiple brain regions, including the hippocampus, whereas reductions in CD200, NGF, and MBP were evident. In a test of spatial learning, PRRSV piglets took longer to acquire the task, had a longer latency to choice, and had a higher total distance moved. Overall, these data demonstrate that viral respiratory infection is associated with a marked increase in activated microglia in the hippocampus, neuroinflammation, and impaired performance in a spatial cognitive task. As respiratory infections are common in human neonates and infants, approaches to regulate microglial cell activity are likely to be important.

Early life iron deficiency impairs spatial cognition in neonatal piglets.

Iron deficiency is common throughout the world and has been linked to cognitive impairments. Using neonatal piglets to model human infants, we assessed the impact of iron deficiency on spatial learning and memory. Artificially reared piglets were fed 1 of 3 liquid diets with varying concentrations of iron: control (CON), mildly deficient (MID), or severely deficient (SID; 100, 25.0, or 10.0 mg iron/kg milk solids, respectively) for 4 wk. Relative to CON, SID and MID piglets had reduced hemoglobin (P < 0.05) as well as magenta skin color (P < 0.001), which correlated with hematocrit (R(2) = 0.76; P < 0.001). SID and MID hemoglobin differed at wk 3 and 4 (P < 0.05). In a hippocampal-dependent, spatial, T-maze task, SID piglets were unable to acquire the task (post hoc contrast: first vs. last day of acquisition), while MID piglets demonstrated deficits in reversal learning (P = 0.032). Iron concentrations in the liver (P < 0.001), serum (P = 0.003), and hippocampus (P = 0.004), but not prefrontal cortex, were lower in MID and SID compared with CON piglets. The level of the transferrin receptor mRNA (TFR) was greater in the prefrontal cortex of CON piglets than in MID and SID piglets (P = 0.001) but not the hippocampus. Gene expression of several neurotrophic factors and proinflammatory cytokines, as well as whole-brain and hippocampal volume, were not affected by dietary treatment. In conclusion, neonatal iron deficiency leads to cognitive impairment, which may be due in part to a reduced iron concentration in the hippocampus.

Place and direction learning in a spatial T-maze task by neonatal piglets.

Pigs are a valuable animal model for studying neurodevelopment in humans due to similarities in brain structure and growth. The development and validation of behavioral tests to assess learning and memory in neonatal piglets are needed. The present study evaluated the capability of 2-week old piglets to acquire a novel place and direction learning spatial T-maze task. Validity of the task was assessed by the administration of scopolamine, an anti-cholinergic drug that acts on the hippocampus and other related structures, to impair spatial memory. During acquisition, piglets were trained to locate a milk reward in a constant place in space, as well as direction (east or west), in a plus-shaped maze using extra-maze visual cues. Following acquisition, reward location was reversed and piglets were re-tested to assess learning and working memory. The performance of control piglets in the maze improved over time (P < 0.0001), reaching performance criterion (80 % correct) on day 5 of acquisition. Correct choices decreased in the reversal phase (P < 0.0001), but improved over time. In a separate study, piglets were injected daily with either phosphate-buffered saline (PBS; control) or scopolamine prior to testing. Piglets administered scopolamine showed impaired performance in the maze compared to controls (P = 0.03), failing to reach performance criterion after 6 days of acquisition testing. Collectively, these data demonstrate that neonatal piglets can be tested in a spatial T-maze task to assess hippocampal-dependent learning and memory.