Early Caffeine Research Articles

Shifting the boundaries for early caffeine initiation in neonatal practice: Results of a prospective, multicenter study on very preterm infants with respiratory distress syndrome.

BackgroundThere is growing evidence that supports the benefits of early use of caffeine in preterm neonates with RDS; however, no formal recommendations specifying the exact timing of therapy initiation have been provided.ObjectivesWe compared neonatal outcomes in infants receiving early (initial dose on the 1st day of life) and late (initial dose on day 2+ of life) caffeine therapy.MethodsUsing data from a prospective, cohort study, we identified 986 infants ≤32 weeks’ gestation with RDS and assessed the timing of caffeine therapy initiation, need for ventilatory support, mortality and incidence of typical complications of prematurity. To adjust for baseline severity, the early and late caffeine groups were propensity score (PS) matched to 286 infants (1:1). Clinical outcomes were compared between the PS-matched groups.ResultsEarly treatment with caffeine citrate was associated with a significantly reduced need for invasive ventilation (71.3% vs 83.2%; P = 0.0165) and total duration of mechanical ventilation (mean 5 ± 11.1 days vs 10.8 ± 14.6 days; P = 0.0000) and significantly lower odds of intraventricular hemorrhage (IVH) (OR 0.4827; 95% CI 0.2999–0.7787) and patent ductus arteriosus (PDA) (OR 0.5686; 95% CI 0.3395–0.9523). The incidence of bronchopulmonary dysplasia (BPD) (36.4% vs 45.8%) and rates of moderate and severe BPD were not significantly different between the two groups. The mortality rates were comparable between the two groups (8.6% vs 8.5%, P = ns)ConclusionEarly caffeine initiation was associated with a decreased need for invasive ventilatory support and lower incidence of IVH and PDA.

Behavioral and pathophysiological outcomes associated with caffeine consumption and repetitive mild traumatic brain injury (RmTBI) in adolescent rats

Given that caffeine consumption is exponentially rising in adolescents and they are at increased risk for repetitive mild traumatic brain injury (RmTBI), we sought to examine the pathophysiological outcomes associated with early life caffeine consumption and RmTBI. Adolescent male and female Sprague Dawley rats received either caffeine in the drinking water or normal water and were then randomly assigned to 3 mild injuries using our lateral impact device or 3 sham procedures. Following injury induction, behavioral outcomes were measured with a test battery designed to examine symptoms consistent with clinical manifestation of PCS (balance and motor coordination, anxiety, short-term working memory, and depressive-like behaviours). In addition, pathophysiological outcomes were examined with histological measures of volume and cellular proliferation in the dentate gyrus, as well as microglia activation in the ventromedial hypothalamus. Finally, modifications to expression of 12 genes (Adora2a, App, Aqp4, Bdnf, Bmal1, Clock, Cry, Gfap, Orx1, Orx2, Per, Tau), in the prefrontal cortex, hippocampus, and/or the hypothalamus were assessed. We found that chronic caffeine consumption in adolescence altered normal developmental trajectories, as well as recovery from RmTBI. Of particular importance, many of the outcomes exhibited sex-dependent responses whereby the sex of the animal modified response to caffeine, RmTBI, and the combination of the two. These results suggest that caffeine consumption in adolescents at high risk for RmTBI should be monitored.

Early Caffeine Prophylaxis and Risk of Failure of Initial Continuous Positive Airway Pressure in Very Low Birth Weight Infants

To test the hypothesis that early caffeine treatment on the day of birth, compared with later treatment in very low birth weight (VLBW, <1500 g) infants receiving continuous positive airway pressure (CPAP) therapy, is associated with a decreased risk of CPAP failure in the first week of life. Multicenter, observational cohort study in 366 US neonatal intensive care units. We evaluated inborn, VLBW infants discharged from 2000 to 2014, who received only CPAP therapy without surfactant treatment on day of life (DOL) 0, had a 5-minute Apgar ≥3, and received caffeine in the first week of life. We used multivariable conditional logistic regression to compare the risk of CPAP failure, defined as invasive mechanical ventilation or surfactant therapy on DOL 1-6, by timing of caffeine treatment as either early (initiation on DOL 0) or routine (initiation on DOL 1-6). We identified 11 133 infants; 4528 (41%) received early caffeine and 6605 (59%) received routine caffeine. Median gestational age was lower in the early caffeine group, 29 weeks (25th, 75th percentiles; 28, 30) vs the routine caffeine group, 30 weeks (29, 31); P < 0.001. The incidence of CPAP failure on DOL 1-6 was similar between the early and routine caffeine groups: 22% vs 21%; adjusted OR = 1.05 (95% CI: 0.93, 1.18). Early caffeine treatment on the day of birth was not associated with a decreased risk of CPAP failure in the first week of life for VLBW infants initially treated with CPAP.

Caffeine ameliorates hyperoxia-induced lung injury by protecting GCH1 function in neonatal rat pups.

BackgroundBronchopulmonary dysplasia (BPD) is a major morbidity in premature infants, and impaired angiogenesis is considered a major contributor to BPD. Early caffeine treatment decreases the incidence of BPD; the mechanism remains incompletely understood.MethodsSprague-Dawley rat pups exposed to normoxia or hyperoxia since birth were treated daily with either 20 mg/kg caffeine or normal saline by intraperitoneal injection from day 2 of life. Lungs were obtained for studies at day 10 and 21.ResultsHyperoxia impaired somatic growth and lung growth in the rat pups. The impaired lung growth during hyperoxia was associated with decreased levels of cyclic AMP (cAMP) and tetrahydrobiopterin (BH4) in the lungs. Early caffeine treatment increased cAMP levels in the lungs of hyperoxia-exposed pups. Caffeine also increased the levels of phosphorylated endothelial nitric oxide synthase (eNOS) at serine1177, total and serine51 phosphorylated GTP-cyclohydrolase-1 (GCH1), and BH4 levels, with improved alveolar structure and angiogenesis in hyperoxia-exposed lungs. Reduced GCH1 levels in hyperoxia were due, in part, to increased degradation by the ubiquitin-proteasome system.ConclusionOur data support the notion that early caffeine treatment can protect immature lungs from hyperoxia-induced damage by improving eNOS activity through increased BH4 bioavailability.

Attenuation of endoplasmic reticulum stress by caffeine ameliorates hyperoxia-induced lung injury.

Rodent pups exposed to hyperoxia develop lung changes similar to bronchopulmonary dysplasia (BPD) in extremely premature infants. Oxidative stress from hyperoxia can injure developing lungs through endoplasmic reticulum (ER) stress. Early caffeine treatment decreases the rate of BPD, but the mechanisms remain unclear. We hypothesized that caffeine attenuates hyperoxia-induced lung injury through its chemical chaperone property. Sprague-Dawley rat pups were raised either in 90 (hyperoxia) or 21% (normoxia) oxygen from postnatal day 1 (P1) to postnatal day 10 (P10) and then recovered in 21% oxygen until P21. Caffeine (20 mg/kg) or normal saline (control) was administered intraperitoneally daily starting from P2. Lungs were inflation-fixed for histology or snap-frozen for immunoblots. Blood caffeine levels were measured in treated pups at euthanasia and were found to be 18.4 ± 4.9 μg/ml. Hyperoxia impaired alveolar formation and increased ER stress markers and downstream effectors; caffeine treatment attenuated these changes at P10. Caffeine also attenuated the hyperoxia-induced activation of cyclooxygenase-2 and markers of apoptosis. In conclusion, hyperoxia-induced alveolar growth impairment is mediated, in part, by ER stress. Early caffeine treatment protects developing lungs from hyperoxia-induced injury by attenuating ER stress.

Association of early versus late caffeine administration on neonatal outcomes in very preterm neonates.

Association of early versus late caffeine administration on neonatal outcomes in very preterm neonates.

Systematic review and meta-analysis of clinical outcomes of early caffeine therapy in preterm neonates.

This study evaluated the therapeutic outcomes of early versus late caffeine therapy in preterm neonates. We performed a systematic literature search in PubMed, Embase, CINAHL and CENTRAL from inception to 30 June 2016 to identify studies investigating the use of early caffeine therapy (initiated at less than 3 days of life) in preterm infants. Effect estimates were combined using random-effects meta-analysis. The primary outcomes for this study were bronchopulmonary dysplasia and mortality. The initial search found 4066 citations, of which 14 studies enrolling a total of 64 438 participants were included. The time of initiation of early caffeine therapy varied from the first 2h to 3 days postnatal. Early caffeine therapy reduced the risk of bronchopulmonary dysplasia in both cohort studies (RR: 0.80, 95% CI: 0.66 to 0.96) and randomized controlled trials (RR: 0.67, 95% CI: 0.56 to 0.81). In cohort studies, neonates treated early with caffeine also showed decreased risks of patent ductus arteriosus, brain injury, retinopathy of prematurity and postnatal steroid use. However, the mortality rate was increased. The findings suggest that early caffeine therapy is associated with reduced incidence of bronchopulmonary dysplasia and may help decrease the burden of morbidities in preterm infants.

Early High-Dose Caffeine Increases Seizure Burden in Extremely Preterm Neonates: A Preliminary Study.

Background: Although evidence suggests that methylxanthines may lower the seizure threshold, the effect of high-dose caffeine on seizure burden in preterm infants is not known. This study reports a secondary post hoc analysis of a randomized controlled trial of early high-dose caffeine citrate therapy in preterm infants, evaluating the effect of caffeine on the seizure burden using amplitude-integrated electroencephalography (aEEG). Methods: Seventy-four preterm infants (≤30 weeks gestation) were randomized to receive high-dose (n = 37, 80 mg/kg over 36 hours) or standard-dose (n = 37, 30 mg/kg over 36 hours) caffeine citrate over the first 36 hours followed by standard maintenance therapy. Simultaneous recording of two-channel amplitude-integrated EEG was conducted over the first 72 hours of life. The primary outcome of this post hoc analysis was cumulative seizure burden over the first 72 hours of life, measured in seconds. Results: Fifteen infants were excluded due to short recordings (≤5 hours) or corrupted data files (n = 7 standard dose; n = 8 high dose). The high-dose caffeine group displayed a trend toward an increased incidence of seizures (40% vs. 58%; p = 0.1) and a threefold increase in seizure duration (48.9 vs. 170.9 seconds; p = 0.1). Conclusion: Early high-dose caffeine therapy was associated with a trend toward an increase in seizure incidence and burden. Future studies of alternative caffeine dosing regimens should include continuous EEG monitoring.

Timing of Caffeine Therapy and Neonatal Outcomes in Preterm Infants: A Retrospective Study.

Background. Caffeine is widely used to treat apnea of prematurity. Here, we evaluated the efficacy of early caffeine (1-2 DOL) in decreasing the incidence of adverse neonatal outcomes. Methods. A retrospective cohort was used to compare the neonatal morbidity of 150 preterm neonates with gestational age ≤29 weeks. Infants were divided into 3 groups based on the initiation timing of caffeine therapy; (1) early caffeine (1-2 DOL), (2) late caffeine (3–7 DOL), and (3) very late caffeine (≥8 DOL). Results. The neonatal outcomes of early caffeine were comparable with those of the late caffeine group. Moreover, when comparing the neonatal morbidity of the very late caffeine group with that of the early caffeine group, multivariable logistic regression analyses were performed. We found that the timing of caffeine did not influence the risk of BPD (OR, 0.393; CI, 0.126–1.223; p = 0.107), but birthweight did (OR, 0.996; CI, 0.993–0.999; p = 0.018) in these infants. Conclusion. Neonatal outcomes of preterm infants were comparable whether caffeine was administered early or late in the first 7 DOL. The risk of BPD in infants receiving caffeine after 8 DOL was irrespective of delayed treatment with caffeine. Our results clearly demonstrate the need for further studies before caffeine prophylaxis can be universally recommended.

Effect of early caffeine on neurodevelopmental outcome of very low-birth weight newborns

Objectives: The objective of this study is to evaluate the effect of early caffeine therapy started within the first 48 h of life on neurodevelopmental outcome in very low birth weight (VLBW) newborns.Study design: VLBW newborns received either caffeine therapy within first 48 h of life (Early group), after 3rd day of life (Late group) or no caffeine during first month of life as per clinical team. A cohort of these newborns (n = 160) who survived were evaluated using Bayley Scale of Infant Development III (BSID III) developmental testing between 18 and 22 months of corrected age.Results: VLBW newborns in the “Early group” had significantly better composite, cognitive, language and motor BSID III scores as compared to those in “Late group” and no caffeine group. Composite BSID III scores were unchanged in the presence or absence of chorioamnionitis for “Early group”, while the BSID III scores were significantly lower in the presence of acute chorioamnionitis in “Late group” and no caffeine group.Conclusions: Early caffeine therapy was associated with better BSID III scores in a cohort of VLBW newborns. Newborns with acute chorioamnionitis benefited from early caffeine therapy.

A pilot randomized trial of high-dose caffeine therapy in preterm infants

BackgroundStandard-dose caffeine improves white matter microstructural development assessed by diffusion MRI. We hypothesized that early high-dose caffeine would result in further improvement in white matter microstructural development.MethodsSeventy-four preterm infants (≤30 weeks gestational age) were randomly assigned to either a high (80 mg/kg IV) or standard (20 mg/kg IV) loading dose of caffeine citrate in the first 24 hours of life. MRI and neurobehavioral testing were undertaken at term equivalent age. Infants returned at 2 years of age for developmental testing.ResultsClinical characteristics were similar between groups, with the exception of higher maternal age in the high-dose caffeine group. There was an increased incidence of cerebellar hemorrhage in infants randomized to high-dose caffeine (36% vs. 10%, p=0.03). Infants in the high-dose caffeine group also demonstrated more hypertonicity (p=0.02) and more deviant neurologic signs (p=0.04) at term equivalent age. Diffusion measures at term equivalent age and developmental outcomes at two years of age did not differ between groups.ConclusionsPreterm infants randomized to early high-dose caffeine had a higher incidence of cerebellar injury with subsequent alterations in early motor performance. The results of this pilot trial discourage a larger randomized controlled trial.

The Timing of In Utero Caffeine Exposure Influences the Morphological, Functional, and Trans‐generational Effects on Adult Hearts

It is recognized that the disruption of the intrauterine environment by nutritional or chemical factors may influence the developing fetus, resulting in long‐term adverse effects in adulthood and subsequent generations. Previously, we demonstrated that in utero caffeine exposure in mice leads to altered cardiac function and morphology in adult offspring, which is accompanied by changes in DNA methylation and cardiac gene expression. We next hypothesized that the timing of in utero caffeine exposure influences which generation is affected, either the exposed embryo (F1) or subsequent generations (F2, exposed gametes, or F3 unexposed). To test this hypothesis, we treated pregnant CD‐1 dams with 20 mg/kg of caffeine daily from E6.5‐9.5, to target DNA re‐methylation in the embryo, or from E10.5‐13.5, to target DNA de‐methylation occurring during gametogenesis. In the F1 generation, early caffeine exposure from E6.5‐9.5 increased LV internal diameter (LVID) and volume, decreased LV posterior wall (LVPW) thickness and decreased % fractional shortening (FS) at 1 year of age, which is consistent with dilated cardiomyopathy. Adult cardiac gene expression was also affected including increased Myh7 expression. For late caffeine exposure from E10.5‐13.5, no effects on cardiac morphology or function were observed at either 12 weeks or 1 year in the F1 generation. However, in the late caffeine exposed F2 generation, we observed increased LVPW thickness, decreased LV volume and increased %FS, which is consistent with concentric cardiac hypertrophy.ConclusionIn utero caffeine exposure leads to changes in adult cardiac morphology and function. The timing of caffeine exposure not only influence which generation is affected but also the cardiac phenotype.

Early Caffeine Use in Very Low Birth Weight Infants and Neonatal Outcomes: A Systematic Review and Meta-Analysis.

The use of caffeine citrate for treatment of apnea in very low birth weight infants showed short-term and long-term benefits. A systematic review and meta-analysis of the literature was undertaken to document the effect providing caffeine early (0-2 days of life) compared to providing caffeine late (≥3 days of life) in very low birth weight infants on several neonatal outcomes, including bronchopulmonary dysplasia (BPD). We searched MEDLINE, the EMBASE database, the Cochrane Library, and KoreaMed for this meta-analysis. The quality of the included studies was assessed using the Newcastle-Ottawa Scale and Jadad's scale. Studies were included if they examined the effect of the early use of caffeine compared with the late use of caffeine. Two reviewers screened the candidate articles and extracted the data from the full-text of all of the included studies. We included a total of 59,136 participants (range 58,997-59,136; variable in one study) from a total of 5 studies. The risk of death (odds ratio [OR], 0.902; 95% confidence interval [CI], 0.828 to 0.983; P=0.019), bronchopulmonary dysplasia (BPD) (OR, 0.507; 95% CI, 0.396 to 0.648; P<0.001), and BPD or death (OR, 0.526; 95% CI, 0.384 to 0.719; P<0.001) were lower in the early caffeine group. Early caffeine use was not associated with a risk of necrotizing enterocolitis (NEC) and NEC requiring surgery. This meta-analysis suggests that early caffeine use has beneficial effects on neonatal outcomes, including mortality and BPD, without increasing the risk of NEC.

Association of early caffeine administration and neonatal outcomes in very preterm neonates.

Advantages of caffeine for apnea of prematurity have prompted clinicians to use it prophylactically even before apnea. To determine the effect of early initiation of caffeine therapy on neonatal outcomes in very preterm infants born in Canada. A retrospective cohort study was conducted. Patients included preterm neonates born at less than 31 weeks' gestation admitted to 29 participating Canadian Neonatal Network neonatal intensive care units between January 1, 2010, and December 31, 2012. Neonates who received caffeine were divided into 2 groups based on the following timing of caffeine initiation: within the first 2 days after birth (early) and on or after the third day following birth (late). A composite of death or bronchopulmonary dysplasia. Of 5517 eligible neonates, 5101 (92.5%) received caffeine (early: 3806 [74.6%]; late: 1295 [25.4%]). There was no difference in weight or gestational age at birth between the groups. Neonates in the early group had decreased odds of a composite outcome of death or bronchopulmonary dysplasia (adjusted odds ratio [AOR], 0.81; 95% CI, 0.67-0.98) and patent ductus arteriosus (AOR, 0.74; 95% CI, 0.62-0.89). There was no difference between the groups in mortality (AOR, 0.98; 95% CI, 0.70-1.37), necrotizing enterocolitis (AOR, 0.88; 95% CI, 0.65-1.20), severe neurological injury (AOR, 0.80; 95% CI, 0.63-1.01), or severe retinopathy of prematurity (AOR, 0.78; 95% CI, 0.56-1.10). In very preterm neonates, early (prophylactic) caffeine use was associated with a reduction in the rates of death or bronchopulmonary dysplasia and patent ductus arteriosus. No adverse impact on any other outcomes was observed.

Early caffeine exposure: transient and long-term consequences on brain excitability.

The influence of pre- and postnatal caffeine treatment on brain excitability during development and adulthood is reviewed. Pre- and postnatal exposure to caffeine induces sex- and age-specific long-term neurochemical alterations in the brain and the behavior of rodents. Because adenosine neuromodulation is closely related to the regulation of brain excitability the increased expression in adenosine receptor system due to neonatal caffeine treatment should cause transient and permanent changes in seizure susceptibility. So far, findings have been focused on primarily developmental changes of the brain adenosine modulatory system and have demonstrated that the alterations are not restricted to a single brain region. Neurobehavioral changes and the anticonvulsant effect of early caffeine exposure are dependent on the caffeine dose, developmental stage of exposure and age of testing. Although outcomes of caffeine treatment are still a matter of debate, our review raise questions concerning the impact of early caffeine treatment on regulation of seizure susceptibility during development and adulthood.

Early caffeine therapy for prevention of bronchopulmonary dysplasia in preterm infants

Objective: To determine if an early commencement of caffeine is associated with improved survival without bronchopulmonary dysplasia (BPD) in preterm infants.Methods: Retrospective data analysis from the Alere Neonatal Database for infants weighing ≤1250 g, and treated with caffeine within the first 10 days of life. The neonatal outcomes were compared between the infants who received early caffeine (0–2 days) with the infants who received delayed caffeine (3–10 days).Results: A total of 2951 infants met the inclusion criteria (early caffeine 1986, late caffeine 965). The early use of caffeine was associated with reduction in BPD (OR 0.69, 95% CI 0.58–0.82, p < 0.001) and BPD or death (OR 0.77, 95% CI 0.63–0.94, p = 0.01). Other respiratory outcomes also improved with the early commencement of caffeine. The frequency of severe intraventricular hemorrhage and patent ductus arteriosus was lower and the length of hospitalization was shorter in infants receiving early caffeine therapy. However, early use of caffeine was associated with an increase in the risk of nectrotizing enterocolits (NEC) (OR 1.41, 95% CI 1.04–1.91, p = 0.027).Conclusion: Early commencement of caffeine was associated with improvement in survival without BPD in preterm infants. The risk of NEC with early caffeine use requires further investigation.

Trends in Caffeine Use and Association between Clinical Outcomes and Timing of Therapy in Very Low Birth Weight Infants

To examine the effect of early initiation of caffeine therapy on neonatal outcomes and characterize the use of caffeine therapy in very low birth weight (VLBW) infants. We analyzed a cohort of 62 056 VLBW infants discharged between 1997 and 2010 who received caffeine therapy. We compared outcomes in infants receiving early caffeine therapy (initial dose before 3 days of life) and those receiving late caffeine therapy (initial dose at or after 3 days of life) through propensity scoring using baseline and early clinical variables. The primary outcome was the association between the timing of caffeine initiation and the incidence of bronchopulmonary dysplasia (BPD) or death. We propensity score-matched 29 070 VLBW infants at a 1:1. Of infants receiving early caffeine therapy, 3681 (27.6%) died or developed BPD, compared with 4591 infants (34.0%) receiving late caffeine therapy (OR, 0.74; 99% CI, 0.69-0.80). Infants receiving early caffeine had a lower incidence of BPD (23.1% vs 30.7%; OR, 0.68; 95% CI, 0.63-0.73) and a higher incidence of death (4.5% vs 3.7%; OR, 1.23; 95% CI, 1.05-1.43). Infants receiving early caffeine therapy had less treatment of patent ductus arteriosus (OR, 0.60; 95% CI, 0.55-0.65) and a shorter duration of mechanical ventilation (mean difference, 6 days; P < .001). Early caffeine initiation is associated with a decreased incidence of BPD. Randomized trials are needed to determine the efficacy and safety of early caffeine prophylaxis in VLBW infants.

Effects of caffeine on cortical epileptic afterdischarges in adult rats are modulated by postnatal treatment

We have previously shown that the effect of acute caffeine injection on cortical epileptic afterdischarges (ADs) in immature rats can be modulated by the postnatal period of repeated caffeine treatment and age of testing during development. To know if these changes persist into adulthood, in the present study adult rats, previously exposed to caffeine from postnatal day 7-11 (10 and 20 mg/kg), and injected with caffeine (10 and 20 mg/kg), respectively, at P67 were compared with groups of naive rats on cortical epileptic ADs. Low-frequency stimulation of sensorimotor cortical area was applied repeatedly with increasing intensity of stimulation current. The acute caffeine injection decreased the thresholds for both the spike-and-wave ADs and accompanying clonic seizures in naive adult rats. In contrast, the acute caffeine administration applied to rats with postnatal caffeine treatment did not change the thresholds for ADs and clonic seizures. In addition, a shorter duration of ADs was registered at some stimulation intensities in rats with early postnatal caffeine administration. Present results demonstrated failure of proconvulsant effects of caffeine after repeated caffeine administration during the early postnatal period persisting up to adulthood.

Early Caffeine Initiation and Neonatal Outcomes Among very Preterm Infants in Canada

Early Caffeine Initiation and Neonatal Outcomes Among very Preterm Infants in Canada

Early caffeine therapy and clinical outcomes in extremely preterm infants

To determine if early caffeine (EC) therapy is associated with decreased bronchopulmonary dysplasia (BPD) or death, decreased treatment of patent ductus arteriosus (PDA), or shortened duration of ventilation. In a retrospective cohort of 140 neonates ≤1250 g at birth, infants receiving EC (initiation <3 days of life) were compared with those receiving late caffeine (LC, initiation ≥3 days of life) using logistic regression. Of infants receiving EC, 25% (21/83) died or developed BPD compared with 53% (30/57) of infants receiving LC (adjusted odds ratio (aOR) 0.26, 95% confidence interval (CI) 0.09 to 0.70; P<0.01). PDA required treatment in 10% of EC infants versus 36% of LC infants (aOR 0.28, 95%CI 0.10 to 0.73; P=0.01). Duration of mechanical ventilation was shorter in infants receiving EC (EC, 6 days; LC, 22 days; P<0.01). Infants receiving EC therapy had improved neonatal outcomes. Further studies are needed to determine if caffeine prophylaxis should be recommended for preterm infants.