Tissue Injury and Astrocytic Reaction, But Not Cognitive Deficits, Are Dependent on Hypoxia Duration in Very Immature Rats Undergoing Neonatal Hypoxia-Ischemia.

Preterm birth and hypoxia-ischemia (HI) are major causes of neonatal death and neurological disabilities in newborns. The widely used preclinical HI model combines carotid occlusion with hypoxia exposure; however, the relationship between different hypoxia exposure periods with brain tissue loss, astrocyte reactivity and behavioral impairments following HI is lacking. Present study evaluated HI-induced behavioral and morphological consequences in rats exposed to different periods of hypoxia at postnatal day 3. Wistar rats of both sexes were assigned into four groups: control group, HI-120 min, HI-180 min and HI-210 min. Neurodevelopmental reflexes, exploratory abilities and cognitive function were assessed. At adulthood, tissue damage and reactive astrogliosis were measured. Animals exposed to HI-180 and HI-210 min had delayed neurodevelopmental reflexes compared to control group. Histological assessment showed tissue loss that was restricted to the ipsilateral hemisphere in lower periods of hypoxia exposure (120 and 180 min) but affected both hemispheres when 210 min was used. Reactive astrogliosis was increased only after 210 min of hypoxia. Interestingly, cognitive deficits were induced regardless the duration of hypoxia and there were correlations between behavioral parameters and cortex, hippocampus and corpus callosum volumes. These results show the duration of hypoxia has a close relationship with astrocytic response and tissue damage progression. Furthermore, the long-lasting cognitive memory deficit and its association with brain structures beyond the hippocampus suggests that complex anatomical changes should be involved in functional alterations taking place as hypoxia duration is increased, even when the cognitive impairment limit is achieved.

Maternal environmental enrichment protects neonatal brains from hypoxic-ischemic challenge by mitigating brain energetic dysfunction and modulating glial cell responses.

There is evidence that maternal milieu and changes in environmental factors during the prenatal period may exert a lasting impact on the brain health of the newborn, even in case of neonatal brain hypoxia-ischemia (HI). The present study aimed to investigate the effects of maternal environmental enrichment (EE) on HI-induced energetic and metabolic failure, along with subsequent neural cell responses in the early postnatal period. Male Wistar pups born to dams exposed to maternal EE or standard conditions (SC) were randomly divided into Sham-SC, HI-SC, Sham-EE, and HI-EE groups. Neonatal HI was induced on postnatal day (PND) 3. The Na+,K+-ATPase activity, mitochondrial function and neuroinflammatory related-proteins were assessed at 24 h and 48 h after HI. MicroPET-FDG scans were used to measure glucose uptake at three time points: 24 h post-HI, PND18, and PND24. Moreover, neuronal preservation and glial cell responses were evaluated at PND18. After HI, animals exposed to maternal EE showed an increase in Na+,K+-ATPase activity, preservation of mitochondrial potential/mass ratio, and a reduction in mitochondrial swelling. Glucose uptake was preserved in HI-EE animals from PND18 onwards. Maternal EE attenuated HI-induced cell degeneration, white matter injury, and reduced astrocyte immunofluorescence. Moreover, the HI-EE group exhibited elevated levels of IL-10 and a reduction in Iba-1 positive cells. Data suggested that the regulation of AKT/ERK1/2 signaling pathways could be involved in the effects of maternal EE. This study evidenced that antenatal environmental stimuli could promote bioenergetic and neural resilience in the offspring against early HI damage, supporting the translational value of pregnancy-focused environmental treatments.

Protective effect of sex steroid hormones on morphological and cellular outcomes after neonatal hypoxia-ischemia: A meta-analysis of preclinical studies.

Sex steroid hormones play an important role in fetal development, brain functioning and neuronal protection. Growing evidence highlights the positive effects of these hormones against brain damage induced by neonatal hypoxia-ischemia (HI). This systematic review with meta-analysis aims to verify the efficacy of sex steroid hormones in preventing HI-induced brain damage in rodent models. The protocol was registered at PROSPERO and a total of 22 articles were included. Moderate to large effects were observed in HI animals treated with sex steroid hormones in reducing cerebral infarction size and cell death, increasing neuronal survival, and mitigating neuroinflammatory responses and astrocyte reactivity. A small effect was evidenced for cognitive function, but no significant effect for motor function; moreover, a high degree of heterogeneity was observed. In summary, data suggest that sex steroid hormones, such as progesterone and 17ß estradiol, improve morphological and cellular outcomes following neonatal HI. Further research is paramount to examine neurological function during HI recovery and standardization of methodological aspects is imperative to reduce the risk of spurious findings.

Effect of environmental enrichment on behavioral and morphological outcomes following neonatal hypoxia-ischemia in rodent models: A systematic review and meta-analysis.

Neonatal hypoxia-ischemia (HI) is a major cause of mortality and morbidity in newborns and, despite recent advances in neonatal intensive care, there is no definitive treatment for this pathology. Once preclinical studies have shown that environmental enrichment (EE) seems to be a promising therapy for children with HI, the present study conducts a systematic review and meta-analysis of articles with EE in HI rodent models focusing on neurodevelopmental reflexes, motor and cognitive function as well as brain damage. The protocol was registered a priori at PROSPERO. The search was conducted in PubMed, Embase and PsycINFO databases, resulting in the inclusion of 22 articles. Interestingly, EE showed a beneficial impact on neurodevelopmental reflexes (SMD= -0.73, CI= [-0.98; -0.47], p< 0.001, I2= 0.0%), motor function (SMD= -0.55, CI= [-0.81; -0.28], p< 0.001, I2= 62.6%), cognitive function (SMD= -0.93, CI= [-1.14; -0.72], p< 0.001, I2= 27.8%) and brain damage (SMD= -0.80, CI= [-1.03; -0.58], p< 0.001, I2= 10.7%). The main factors that potentiate EE positive effects were enhanced study quality, earlier age at injury as well as earlier start and longer duration of EE exposure. Overall, EE was able to counteract the behavioral and histological damage induced by the lesion, being a promising therapeutic strategy for HI.

Differential Age-Dependent Mitochondrial Dysfunction, Oxidative Stress, and Apoptosis Induced by Neonatal Hypoxia-Ischemia in the Immature Rat Brain.

Neonatal hypoxia-ischemia (HI) is among the main causes of mortality and morbidity in newborns. Experimental studies show that the immature rat brain is less susceptible to HI injury, suggesting that changes that occur during the first days of life drastically alter its susceptibility. Among the main developmental changes observed is the mitochondrial function, namely, the tricarboxylic acid (TCA) cycle and respiratory complex (RC) activities. Therefore, in the present study, we investigated the influence of neonatal HI on mitochondrial functions, redox homeostasis, and cell damage at different postnatal ages in the hippocampus of neonate rats. For this purpose, animals were divided into four groups: sham postnatal day 3 (ShP3), HIP3, ShP11, and HIP11. We initially observed increased apoptosis in the HIP11 group only, indicating a higher susceptibility of these animals to brain injury. Mitochondrial damage, as determined by flow cytometry showing mitochondrial swelling and loss of mitochondrial membrane potential, was also demonstrated only in the HIP11 group. This was consistent with the decreased mitochondrial oxygen consumption, reduced TCA cycle enzymes, and RC activities and induction of oxidative stress in this group of animals. Considering that HIP3 and the sham animals showed no alteration of mitochondrial functions, redox homeostasis, and showed no apoptosis, our data suggest an age-dependent vulnerability of the hippocampus to hypoxia-ischemia. The present results highlight age-dependent metabolic differences in the brain of neonate rats submitted to HI indicating that different treatments might be needed for HI newborns with different gestational ages.

Differential glucose and beta-hydroxybutyrate metabolism confers an intrinsic neuroprotection to the immature brain in a rat model of neonatal hypoxia ischemia.

Neonatal hypoxia ischemia (HI) is the main cause of newborn mortality and morbidity. Preclinical studies have shown that the immature rat brain is more resilient to HI injury, suggesting innate mechanisms of neuroprotection. During neonatal period brain metabolism experience changes that might greatly affect the outcome of HI injury. Therefore, the aim of the present study was to investigate how changes in brain metabolism interfere with HI outcome in different stages of CNS development. For this purpose, animals were divided into 6 groups: HIP3, HIP7 and HIP11 (HI performed at postnatal days 3, 7 and 11, respectively), and their respective shams. In vivo [18F]FDG micro positron emission tomography (microPET) imaging was performed 24 and 72 h after HI, as well as ex-vivo assessments of glucose and beta-hydroxybutyrate (BHB) oxidation. At adulthood behavioral tests and histology were performed. Behavioral and histological analysis showed greater impairments in HIP11 animals, while HIP3 rats were not affected. Changes in [18F]FDG metabolism were found only in the lesion area of HIP11, where a substantial hypometabolism was detected. Furthermore, [18F]FDG hypometabolism predicted impaired cognition and worst histological outcomes at adulthood. Finally, substrate oxidation assessments showed that glucose oxidation remained unaltered and higher level of BHB oxidation found in P3 animals, suggesting a more resilient metabolism. Overall, present results show [18F]FDG microPET predicts long-term injury outcome and suggests that higher BHB utilization is one of the mechanisms that confer the intrinsic neuroprotection to the immature brain and should be explored as a therapeutic target for treatment of HI.

Enriched experience during pregnancy and lactation protects against motor impairments induced by neonatal hypoxia-ischemia.

Neonatal hypoxia-ischemia (HI) is responsible for movement disorders in preterm infants. Non-pharmacological strategies, such as environmental enrichment (EE) during adulthood, have shown positive effects on promoting sensorimotor recovery after HI. However, little is known about the effects of perinatal EE on sensorimotor function following HI. In present study we investigated the hypothesis that enriched experiences during pregnancy and lactation would reduce motor impairments caused by a model of neonatal HI in rats. At postnatal day (PND) 3, Wistar pups of both sexes were subject to the modified Rice-Vannucci model. Motor function was evaluated from PND 60 to PND 64. HI caused a reduction in the forepaws strength and worsening of movement quality in the right forepaw. These effects were attenuated in animals receiving prenatal or lactational EE, which showed better performance when compared to the control group. Moreover, enriched experiences during lactation reversed HI-induced asymmetric use of the forepaws and the trend to increased paw errors in a walking test. Lower scores were found in the contralateral forepaw placement in HI animals, except when EE was provided at both stages of neurodevelopment. These results indicate that enriched experiences reduce motor impairments, i.e, measured in force, asymmetry and coordination domains, and that EE during lactation is more effective in promoting post-injury recovery. These data support that early therapeutic interventions might enhance functional reorganization at a period of high brain plasticity and that enriched-like experience might be encouraged in pediatric rehabilitation programs, in order to reduce long-term movement disorders after neonatal brain insults.

Preventive and therapeutic effects of environmental enrichment in Wistar rats submitted to neonatal hypoxia-ischemia.

Environmental enrichment (EE) at early stages of neurodevelopment attenuates HI-induced behavioral, histological and cellular damage. However, the effects of EE exposure during gestational or early postnatal period and the possible influence of sexual dimorphism on EE protection are not fully understood. Present study evaluated the effects of pre-natal and postnatal EE, as well as their combination, in male and female rats submitted to neonatal HI at postnatal day (PND) 3. Wistar rats were housed in EE or in standard condition (SC) during all pregnancy. At PND1, the litters were randomly allocated to the same prenatal environment during lactation (SC + SC or EE + EE) or housed in a new environment until weaning (SC + EE or EE + SC). Behavioral tasks were performed from PND 60-75. Then, animals were euthanized for biochemical and histological analysis. Prenatal and early postnatal EE alone improved performance of HI males in the Water Maze spatial memory task, while HI females were most benefited from early postnatal stimulation. Moreover, EE attenuated HI-induced lower anxiety-like behavior in rats of both sexes and decreased hyperlocomotion in HI females. Hippocampus tissue preservation and higher VEGF and TrkB levels were observed in all HI groups exposed to EE. Interestingly, HI males exposed to prenatal or postnatal EE alone exhibited higher GFAP levels and additional tissue preservation. Therefore, both prenatal and early postnatal environmental enrichment cause attenuation of HI-induced impairments, revealing their preventive and therapeutic actions, possibly due to VEGF and astrocyte activity; some of these effects are sex-specific.

Pregnancy as a valuable period for preventing hypoxia-ischemia brain damage.

Neonatal brain Hypoxia-Ischemia (HI) is one of the major causes of infant mortality and lifelong neurological disabilities. The knowledge about the physiopathological mechanisms involved in HI lesion have increased in recent years, however these findings have not been translated into clinical practice. Current therapeutic approaches remain limited; hypothermia, used only in term or near-term infants, is the golden standard. Epidemiological evidence shows a link between adverse prenatal conditions and increased risk for diseases, health problems, and psychological outcomes later in life, what makes pregnancy a relevant period for preventing future brain injury. Here, we review experimental literature regarding preventive interventions used during pregnancy, i.e., previous to the HI injury, encompassing pharmacological, nutritional and/or behavioral strategies. Literature review used PubMed database. A total of forty one studies reported protective properties of maternal treatments preventing perinatal hypoxia-ischemia injury in rodents. Pharmacological agents and dietary supplementation showed mainly anti-excitotoxicity, anti-oxidant or anti-apoptotic properties. Interestingly, maternal preconditioning, physical exercise and environmental enrichment seem to engage the same referred mechanisms in order to protect neonatal brain against injury. This construct must be challenged by further studies to clearly define the main mechanisms responsible for neuroprotection to be explored in experimental context, as well as to test their potential in clinical settings.

Locomotor Training Promotes Time-dependent Functional Recovery after Experimental Spinal Cord Contusion.

Locomotor training (LT) has been exhaustively investigated as a treatment for the spinal cord injury (SCI), however the literature reports both positive and negative effects over the functional recovery. The initiation period of LT following SCI is one of the major variables that needs attention. To investigate the better period, three different starting times were investigated after SCI in rats. Methods: Wistar rats were randomly divided into groups: control, SCI (rats with spinal cord contusion), and SCI groups exposed to LT starting 7, 14 or 28 days after the injury (SCI-T7, SCI-T14 and SCI-T28). LT was performed on a treadmill, five days a week, 20 minutes per day, for ten weeks. Basso, Breattie and Bresnahan (BBB) scale and Horizontal Ladder walking test were used to evaluate the motor function; at the end, morphological and biochemical analyses of the spinal cords, tibialis anterior and soleus muscles were performed. Results: SCI-T14 and SCI-T28 groups had an improvement in both behavioral tests, while SCI-T7 presented a worsening in the functional performance. Late training groups preserved motoneurons in the spinal cord, showed larger muscle fiber areas and higher BDNF expression in tibialis anterior muscle. SCI-T7 group had higher lesion volume after LT in comparison with the SCI group. Late onset of LT promoted an increment of the hindlimb function, while early onset of training worsened the functional recovery of the SCI animals. These results demonstrate a critical LT starting time after the injury, contributing to define the best therapeutic window for rehabilitation.

Prenatal and Early Postnatal Environmental Enrichment Reduce Acute Cell Death and Prevent Neurodevelopment and Memory Impairments in Rats Submitted to Neonatal Hypoxia Ischemia.

Environmental enrichment (EE) is an experimental strategy to attenuate the negative effects of different neurological conditions including neonatal hypoxia ischemia encephalopathy (HIE). The aim of the present study was to investigate the influence of prenatal and early postnatal EE in animals submitted to neonatal HIE model at postnatal day (PND) 3. Wistar rats were housed in EE or standard conditions (SC) during pregnancy and lactation periods. Pups of both sexes were assigned to one of four experimental groups, considering the early environmental conditions and the injury: SC-Sham, SC-HIE, EE-sham, and EE-HIE. The offspring were euthanized at two different time points: 48 h after HIE for biochemical analyses or at PND 67 for histological analyses. Behavioral tests were performed at PND 7, 14, 21, and 60. Offspring from EE mothers had better performance in neurodevelopmental and spatial memory tests when compared to the SC groups. HIE animals showed a reduction of IGF-1 and VEGF in the parietal cortex, but no differences in BDNF and TrkB levels were found. EE-HIE animals showed reduction in cell death, lower astrocyte reactivity, and an increase in AKTp levels in the hippocampus and parietal cortex. In addition, the EE was also able to prevent the hippocampus tissue loss. Altogether, present findings point to the protective potential of the prenatal and early postnatal EE in attenuating molecular and histological damage, as well as the neurodevelopmental impairments and the cognitive deficit, caused by HIE insult at PND 3.

Longer hypoxia-ischemia periods to neonatal rats causes motor impairments and muscular changes.

Prematurity and hypoxia-ischemia (HI) can lead to movement disorders in infants. Considering that mild-moderate HI induced at postnatal day (PND) 3 has failed to produce motor disabilities similar to those seen in pre-term newborns, the main goal of the present study was to verify whether longer hypoxia periods would mimic motor function impairment, brain and muscle morphological alterations. Forty-nine Wistar rat pups of both sexes were randomly assigned to surgical control (CG) and HI groups. HI animals were submitted to the Levine-Rice model at PND 3, and exposed to 120 (HI-120'), 180 (HI-180') or 210 (HI-210') minutes of hypoxia (FiO2: 0.08). Sensorimotor function was assessed as from PND 35-45, by means of grasping strength, adhesive removal, cylinder and ladder walking tests. Histological staining was used to quantify the striatal volume and the cross-sectional area (CSA) of skeletal muscles. Cylinder and adhesive removal test evidenced that HI-180' and HI-210' groups had asymmetrical use of the forepaws when compared to controls. HI animals showed a decrease in the step placement quality and an increase in step errors when compared to CG (P⩽0.05). Reduction in striatal volume correlates with behavioral assessment, HI-180' and HI-210' groups presented lower biceps brachii and tibialis anterior CSA. These results show that rats exposed to longer hypoxic periods at PND3 have encephalic and sensorimotor impairments that mimic those observed in preterm infants. Morphological changes in muscle tissue evidence a new pathophysiological characteristic of the HI model that might be of relevance for the study of sensorimotor deficits.

Galantamine administration reduces reactive astrogliosis and upregulates the anti-oxidant enzyme catalase in rats submitted to neonatal hypoxia ischemia.

Neonatal hypoxia ischemia (HI) plays a role in the etiology of several neurological pathologies and causes severe sequelae. Acetylcholine is a neurotransmitter in the central nervous system and cholinesterase inhibitors have demonstrated a positive action over HI induced deficits. In order to evaluate the effects of pre and post-hypoxia administrations of galantamine, a cholinesterase inhibitor, in a model of perinatal HI, Wistar rats in the post-natal day 7 (PND7) were subjected to a combination of unilateral occlusion of the right carotid artery with the exposure to a 1h hypoxia. Intraperitoneal injections of galantamine were administered in two different protocols: one pre and other post-hypoxia. The analysis of brain structures volume at PND45 showed that pre-hypoxia galantamine treatment prevented tissue injury to the ipsilesional hippocampus. Also, immunofluorescence showed HI-induced increase in the number of astrocytes that was prevented by pre-hypoxia treatment. Biochemical analysis was performed in the ipsilesional hippocampus at PND8 and revealed that pre-hypoxia galantamine treatment: 1) prevented the neuronal loss induced by HI; 2) reduced the HI-induced hypertrophy of astrocytes; and 3) caused an increase in the activity of the anti-oxidant enzyme catalase. Overall, treatment with galantamine was able to prevent the brain damage, increase the survival of neurons, reduce astrocytic reaction and increase the activity of the anti-oxidant enzyme catalase in rats submitted to neonatal hypoxia ischemia.