ORIGINAL RESEARCH published: 13 February 2019 doi: 10.3389/fnbeh.2019.00007 Severe Perinatal Hypoxic-Ischemic Brain Injury Induces Long-Term Sensorimotor Deficits, Anxiety-Like Behaviors and Cognitive Impairment in a Sex-, Age- and Task-Selective Manner in C57BL/6 Mice but Can Be Modulated by Neonatal Handling Aida Muntsant 1,2 , Kalpana Shrivastava 2,3 , Mireia Recasens 2,3 and Lydia Giménez-Llort 1,2 * 1 Department of Psychiatry and Forensic Medicine, School of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Department of Cell Biology, Physiology & Immunology, Universitat Autònoma de Barcelona, Barcelona, Spain Perinatal brain injury (PBI) leads to neurological disabilities throughout life, from motor deficits, cognitive limitations to severe cerebral palsy. Yet, perinatal brain damage has Edited by: limited therapeutic outcomes. Besides, the immature brain of premature children is Rosario Montirosso, at increased risk of hypoxic/ischemic (HI) injury, with males being more susceptible Eugenio Medea (IRCCS), Italy to it and less responsive to protective/therapeutical interventions. Here, we model in Reviewed by: Michael Arthur Van Der Kooij, male and female C57BL/6 mice, the impact of neonatal HI and the protective effects Johannes Gutenberg University of neonatal handling (NH), an early life tactile and proprioceptive sensory stimulation. Mainz, Germany From postnatal day 1 (PND1, modeling pre-term) to PND21 randomized litters received Sharon Casavant, University of Connecticut, either NH or left undisturbed. HI brain damage occurred by permanent left carotid United States occlusion followed by hypoxia at PND7 (modeling full-term) in half of the animals. The *Correspondence: behavioral and functional screening of the pups at weaning (PND23) and their long-term Lydia Giménez-Llort outcomes (adulthood, PND70) were evaluated in a longitudinal study, as follows: lidia.gimenez@uab.cat somatic development (weight), sensorimotor functions (reflexes, rods and hanger Received: 21 November 2018 tests), exploration [activity (ACT) and open-field (OF) test], emotional and anxiety-like Accepted: 09 January 2019 behaviors [corner, open-field and dark-light box (DLB) tests], learning and memory [T- Published: 13 February 2019 maze (TM) and Morris Water-Maze (MWM)]. HI induced similar brain damage in both Citation: Muntsant A, Shrivastava K, sexes but affected motor development, sensorimotor functions, induced hyperactivity Recasens M and Giménez-Llort L at weaning, and anxiety-like behaviors and cognitive deficits at adulthood, in a sex- (2019) Severe Perinatal Hypoxic-Ischemic Brain Injury and age-dependent manner. Thus, during ontogeny, HI affected equilibrium especially Induces Long-Term Sensorimotor in females and prehensility in males, but only reflexes at adulthood. Hyperactivity of Deficits, Anxiety-Like Behaviors and HI males was normalized at adulthood. HI increased neophobia and other anxiety-like Cognitive Impairment in a Sex-, Age- and Task-Selective Manner in behaviors in males but emotionality in females. Both sexes showed worse short/long- C57BL/6 Mice but Can Be term learning, but memory was more affected in males. Striking neuroprotective Modulated by Neonatal Handling. Front. Behav. Neurosci. 13:7. effects of NH were found, with significantly lower injury scores, mostly in HI males. doi: 10.3389/fnbeh.2019.00007 At the functional level, NH reversed the impaired reflex responses and improved Frontiers in Behavioral Neuroscience | www.frontiersin.org 1 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury memory performances in hippocampal-dependent spatial-learning tasks, especially in males. Finally, neuropathological correlates referred to atrophy, neuronal densities and cellularity in the affected areas [hippocampal-CA, caudate/putamen, thalamus, neocortex and corpus callosum (CC)] point out distinct neuronal substrates underlying the sex- and age- functional impacts of these risk/protection interventions on sensorimotor, behavioral and cognitive outcomes from ontogeny to adulthood. Keywords: neonatal hypoxic ischemic injury, neonatal handling, sensory stimulation, animal model, sex, gender medicine, behavior, cognition INTRODUCTION adult stroke or after neonatal HI had been documented (Bona et al., 1998; Hagberg et al., 2004; Hurn et al., 2005; Smith Perinatal brain injury (PBI) due to hypoxia-ischemia (HI) is et al., 2014; Netto et al., 2017). These differences are justified such a devastating early insult that it is considered to be a by the presence of sex-specific hormones that may influence the major contributor to perinatal morbidity and mortality. The consequences of early HI brain injury (Hill and Fitch, 2012) prevalence of neonatal HI encephalopathy (HIE) due to an but also to socio-economic and neonatal variables (Månsson oxygen and glucose deprivation during birth is 1.5–3 and et al., 2015). In spite of these sex differences, most studies up to 6 per 1,000 livebirths in developed and developing involving HI still demonstrate usage of male and female animals countries, respectively (Kurinczuk et al., 2010). The perinatal indistinctly (i.e., Chou et al., 2001; Ten et al., 2003; Lubics brain is highly susceptible to damage due to the prevailing et al., 2005; Spandou et al., 2005; Ikeda et al., 2006; Pazos et al., development processes. The most vulnerable regions to injury 2012). are the ones with greatest metabolic demands (sensorimotor On the other hand, developmental psychobiology and cortex and basal ganglia, thalamus, cerebellum and brainstem; neuroscience have pointed out ontogeny as a singular window Thorngren-Jerneck et al., 2001). Consequently, PBI can lead of brain vulnerability but also plasticity, where early-life to long-term neurologic disability in both children and paradigms involving exposure to distinct stimuli during the adults, including cognitive limitations, learning difficulties, 1st weeks of life are shown to be critical for short- and attention or motor deficits (van Handel et al., 2007) and long-lasting modeling of the brain structure and function even cerebral palsy, and seizures (Platt et al., 2007). Despite (Levine, 1957; Levine and Broadhurst, 1963; Levine et al., the improvements in neonatal care, brain damage in term 1967). In rodents, early postnatal stimulation [neonatal handling newborn infants still remains a clinical problem, with research (NH) in its most frequent form] consisting of brief maternal constrained by obvious ethical limitations. Most importantly, separation with/without tactile stimulation, prompts profound the immature brain of premature children is at increased risk and long-lasting effects of anxiety and stress responses, novelty- of hypoxic ischemic injury (Vannucci and Hagberg, 2004) with seeking, learning and memory through different epigenetic males born prematurely being reported as most susceptible neurobiological mechanisms (revised by Fernández-Teruel et al., to it and with worse developmental and adult outcomes 2002). More importantly, compelling effects of this intervention (Elsmén et al., 2004; Peacock et al., 2012; Månsson et al., also include the rescue of perinatal brain insults due to stress, 2015). malnutrition or alcohol exposure (revised by Raineki et al., It is considered that the experimental model of HI-induced 2014). In the case of brain injury induced by HI, prevention of neonatal injury initially described by Vannucci and Vannucci hippocampal damage (Rodrigues et al., 2004) and improvement (1997) for the rat (Rice et al., 1981), and also adapted to the of learning (Chou et al., 2001) were elicited in rats by maternal mouse in several laboratories (Sheldon et al., 1998; Hagberg separation and tactile stimulation starting at PND8. Beneficial et al., 2002; Northington, 2006) is a useful translational technique effects of tactile sensory stimulation have been attributed to the to better understand the effects of HI injury in human brain. fact that neural pathways from skin to the CNS mature before Moreover, this technique at postnatal day (PND) 7–10 is other sensory systems (Montagu, 1953). equivalent to a term human infant (Semple et al., 2013; Mallard Therefore, the aim of the current research work was to and Vexler, 2015). Although many experimental studies, mostly do a longitudinal study of the behavioral and functional in rats, report morphological, biophysical and biochemical impact of HI brain injury in male and female mice, at changes following HI brain insult (Towfighi et al., 1991; Huang weaning and at adulthood. We also aimed to assess the effects and Castillo, 2008; Shrivastava et al., 2012), there is a scarcity of NH used as a protective sensory intervention. For this of data to understand the consequential behavioral changes. purpose, we evaluated the short- and long-term effects of a However, some reports do suggest that injured animals exhibited neonatal HI insult in the behavioral profile of gold-standard sensorimotor deficits (i.e., Jansen and Low, 1996; Bona et al., C57BL/6 mice strain at PND7, an age modeling full-term 1997) and suffered certain learning disabilities (i.e., Young et al., babies. At the same time, a group of animals was used to 1986; Balduini et al., 2000; Ikeda et al., 2001; McAuliffe et al., assess the potential preventive effects of NH administered 2006). Sex differences regarding the final outcome after an during the ontogenic development of the pups, from PND1 to Frontiers in Behavioral Neuroscience | www.frontiersin.org 2 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury weaning (PND21), mimicking an intervention started in for another 3–4 s, stroked again and then returned to its home pre-term babies and lasting all through their childhood. For cage. When all the pups from 1 L were back in their home cage, the behavioral and functional screening of the pups at weaning the mother was returned to it. The same procedure (without (PND23) and their long-term outcomes (adulthood, PND70) weighing the animals) was conducted in the afternoon (2nd we used a series of tests to evaluate four dimensional areas: time; approximately at 4:30 p.m.). NH finished at PND 21. somatic development and sensorimotor integration, exploratory Weaning was done at PND 21, after finishing the last NH session. behavior, emotionality and anxiety-like behaviors, and cognition. Non-handled groups were left undisturbed, except for regular Finally brain pathology was measured by means of brain injury cage cleaning once a week, until weaning. score to evaluate the neurologic impact of the risk/protection events in different neuroanatomical regions at 90 days of Behavioral and Functional Assessments age (PND90). This will allow us to find the functional Animals were assessed in a longitudinal design, at weaning correlates. (PND23) and adulthood (PND70 or 70-days-old) as summarized in the time line shown in Figure 1. A three-stage protocol (Figure 1B) for behavioral and functional phenotype assessment MATERIALS AND METHODS (Giménez-Llort et al., 2002) evaluated the somatic development (primary screening), sensorimotor functions and motor activity Animals (secondary screening) as well as non-cognitive and cognitive Sixty-six C57BL/6 mice were bred and maintained in macrolon functions (tertiary screening). The tests used were as follows: cages (35 cm × 35 cm × 25 cm) under standard laboratory somatic development (weight), sensorimotor functions [visual conditions (food and water ad libitum, 22◦ C ± 2◦ C, 50%–70% and hind limb reflexes, rod test and hanger test, cylinder test relative humidity, 12 h light:dark cycle, lights on at 08:00 h). (CYT)], locomotor [activity (ACT) and open-field (OF) test] as well as non-cognitive and cognitive functions-emotional and Experimental Design anxiety-like behaviors [corner, OF and dark-light box tests A longitudinal study with factorial design HI × NH × S was (DLB)]; learning and memory (TM test and MWM). All the made to evaluate the functional impact of HI, the effects of apparatus were thoroughly cleaned, with 5% ethanol, and dried NH in sham and HI animals, and the factors sex and age. between trials/animals. Age factor was studied to evaluate the long-term effects, from weaning at PND23 to adulthood (PND70), of the risk/protective Body Weight (BW) interventions studied. The total 66 mice came from 12 L Body Weight (BW) was monitored at weaning (PND23), with an average of 5.50 + 0.45 pups. Litters were randomly adulthood (PND70) and at the end point (PND90). distributed by treatments and gender in 8 different experimental groups (n = 7–11, Figure 1A). Two animals (1 male/sham, Sensorimotor Functions (SMT) 1 female/hypoxia) died from PND23 to PND90, and were The physical condition of the mice was evaluated by their BW therefore excluded of the statistical analysis. and performance in sensorimotor tasks. Visual reflex and hind limbs extension reflex were measured three times by holding the Hypoxia/Ischemia animal by its tail and slowly lowering it toward a black surface. HI brain damage occurred at PND7 by permanent left carotid Complete extension of the forelimbs towards the surface (visual occlusion and exposure to hypoxia as previously described reflex) or the extension of hind limbs were considered a positive (Sheldon et al., 2001). Briefly, a midline ventral skin incision response. Motor coordination and equilibrium were assessed was made under isoflurane anesthesia (4.5% v/v for induction twice (20-s trials) in two consecutive rod tasks of increasing and 2.5% v/v for maintenance, and 0.6 L/min of O2 ); the left difficulty. The distance covered and the latency to fall off a 1.3 cm carotid artery was exposed and sutured with a 8/0 silk surgical wide wooden wire rod and a 1 cm diameter metal wire rod (both, suture. After surgery, pups were returned to their dam for at least 1 m long) were recorded. The hanger test was used to measure 1.5 h to recover. Later, litters were placed for 55 min in a hypoxic prehensility or grasping and motor coordination by the distance chamber containing 8% of oxygen balanced with nitrogen, with covered and the number of elements of support and the latency controlled humidity and temperature maintained at 37◦ C. to fall. The animal was allowed to cling with its forepaws from the middle of a horizontal wire (2 mm diameter, 40 cm length, Handling divided into eight 5 cm segments) for two trials of 5 s. A third NH was administered from PNDs 1 to 21 (Fernández-Teruel trial of 60 s was used to complement these measures with that of et al., 1991). The first daily session, administered in the morning muscle strength or resistance. All the apparatus were suspended (9:30 a.m.), consisted of first removing the mother from the 40 cm above a padded table. litter, and then weighing the pups and placing them gently and individually in plastic cages (35 cm × 15 cm × 25 cm) lined Cylinder Test (CYT) with soft article towel. After 4 min in this situation, each pup The CYT was used to assess forelimb asymmetry (Schallert was individually (and gently) handled and stroked thrice with et al., 2000). Animals were individually placed in a Plexiglas the thumb on the dorsal surface (rostro-caudal direction) for transparent cylinder (10 cm diameter, 12 cm height). Each animal 3–4 s and returned to the same cage for the remaining 4 min. was video-recorded for 5 min. Initial forepaw (left/right/both) At the end of the 8-min period, each pup was gently handled placement of each weight-bearing full rear was recorded. Frontiers in Behavioral Neuroscience | www.frontiersin.org 3 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury FIGURE 1 | (A) Time line of the experimental schedule. (B) Behavioral and functional phenotype assessment: a three-stage protocol for behavioral and functional phenotype assessment evaluated the somatic development (primary screening), sensorimotor functions and motor activity (secondary screening) as well as non-cognitive and cognitive functions (tertiary screening). The tests used were the following: somatic development (weight), sensorimotor functions (visual and hind limb reflexes, rod test and hanger test), locomotor [activity test (ACT) and open-field (OF) test] as well as non-cognitive and cognitive functions-emotional and anxiety-like behaviors [corner, OF and dark-light box (DLB) tests]; learning and memory [T-maze test (TM) and Morris Water Maze (MWM)]. The behavioral assessment was performed at weaning and/or adulthood. The asymmetry score that reflected the preference of the × 25 cm—simultaneously, Sensor Unit PANLAB 0603, Panlab, unimpaired (left) limb was calculated according to the following S.L., Barcelona, Spain) set to measure horizontal and vertical formula:[(number of left contacts + 12 both contacts)/number spontaneous motor activity during 30 min. Animals were of (left + right + both contacts)]×100; where 50% indicates individually tested in a standard (but novel and clean) home cage an animal that explores symmetrically with both limbs, higher containing a small amount of clean sawdust on the floor. scores (>50%) indicate a greater reliance on the ipsilateral limb, Corner Test (CT) and Open Field Test (OF) and lower scores (<50%) indicate a greater reliance on the Neophobia was assessed in the corner test (CT) for 30 s. Animals contralateral limb. We also measured the number and the total were individually placed in the center of a clean standard amount of grooming time, the latency of ‘‘hoppy’’ or ‘‘pop-corn home cage, filled with wood save bedding. Latency of the first behavior’’ described by Wahlsten (1974) as vigorous jumping rearing, number of corners visited and of vertical displacements shown in pups, and the number of full rears performed in the (rearings) were recorded. Immediately after, exploratory and 5 min. anxiety-like behaviors were measured during 5 min in a white open-field (homemade, wooden, 55 × 55 × 25 cm) under 20 lux Spontaneous Motor Activity Test (ACT) light conditions. Mice were individually placed in the center The mice were individually tested in a multicage activity of the arena. Horizontal (crossings, 10 × 10 cm) and vertical meter system (four home cages −35 cm × 35 cm (rearings) activities were recorded for each minute of the test. Frontiers in Behavioral Neuroscience | www.frontiersin.org 4 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury Dark–Light Box Test (DLB) hidden platform, which was now located in the middle of the S Anxiety and risk assessment were measured for 5 min after quadrant. Mice that failed to find the platform within 60 s were introducing the animals into the dark compartment of the DLB placed on it for 10 s, the same period as was allowed for the (Panlab, S.L., Barcelona, Spain). The apparatus consisted of successful animals. White geometric figures, one hung on each two compartments (black, 27 cm × 18 cm × 27 cm, white, wall of the room, were used as external visual clues. 27 cm × 27 cm × 27 cm illuminated by a red 20 W bulb) connected by an opening (7 cm × 7 cm). The experimental Removal room was kept in darkness (without illumination). Mice were Two hours after the last trial of the place learning task, the introduced into the black compartment and observed for 5 min. platform was removed from the maze and the mice performed Total number of entries (all four paws), and time spent in the a probe trial of 60 s to evaluate their spatial memory for the white-illuminated compartment were recorded. Latency to enter, platform position. time spent and number of entries into the lit compartment, Analyses the number of stretch attendances and self-groomings were Behavior was evaluated by both direct observation and recorded. analysis of videotape-recorded images. Variables of time (escape latency, quadrant preference), distance covered, and swimming T-Maze Test (TM) speed were analyzed in all the trials of the tasks. The Exploratory activity, anxiety, working and short-term memory escape latency was readily measured with a stopwatch by an were assessed in an enclosed TM (woodwork; short arm: observer who was unaware of the experimental group, and 40 × 15 × 30; goal arms: 30 × 15 × 30 cm), three trials 15 min was confirmed during the subsequent video-tracking analysis. apart, in a day. Each trial involved one forced and one free choice. A video camera placed above the water maze recorded Mice were placed inside the short arm of the maze and the the animal’s behavior and thereafter an automated system latency to reach the intersection and the time elapsed until mice (Smart, Panlab S.L., Barcelona, Spain) enabled computerized completed 20 s in the forced arm were recorded. Fifteen seconds measurement of the distance traveled by the animal during later, mice were allowed to explore the maze in a free choice trial the trials. The swimming speed (cm/s) of the mice during where both arms were accessible. The arm chosen was recorded each trial was calculated. In the probe trial, the time and considered an error if it was not different of that in the forced spent in each of the four quadrants, the distance traveled choice. along them, and the number of crossings over the removed Morris Water Maze (MWM) platform position (annulus crossings) were also measured Animals were tested for spatial learning and memory in three retrospectively by means of the automated video-tracking paradigms in the MWM test consisting of 1 day of cue learning analysis. and 4 days of place learning for spatial reference memory, followed by one probe trial. Mice were trained to locate a hidden Neuropathological Analysis Brain damage was analyzed by histological analysis at platform (7 cm diameter, 1 cm below the water surface) in a PND90 (Shrivastava et al., 2012). Mice were i.p. anesthetized circular pool for mice (Intex Recreation Corp., Long Beach, CA, (ketamine and xylazine 80/10 mg/Kg) and perfused using USA; 91 cm diameter, 40 cm height, 25◦ C opaque water), located 4% paraformaldehyde in phosphate buffer (PB, pH 7.4). in a completely black painted 6 m2 test room. Mice failing to find Subsequently, brains were postfixed for 4 h in the same the platform were placed on it for 10 s, the same period as the fixative, cryoprotected in 30% sucrose, frozen with dry CO2 , successful animals. The protocol (Giménez-Llort et al., 2007) was and finally stored at −80◦ C until use. Brains were serially used as follows: 1 day of cue learning, 4 days of place learning cut in a cryostat (Leica CM3050 S) in 30 µm thick sections followed by a probe trial. and stored in −20◦ C mounted on Flex IHC slides (Dako). Cue Learning With a Visible Platform To determine the injury score, slides were processed for Nissl On the first day, the animals were tested for the cue learning staining. One series of parallel sections from each animal of a visual platform consisting of four trials in 1 day. In each (6–10 mice/survival time) was air dried at room temperature trial, the mouse was gently released (facing the wall) from one for an hour, rinsed and incubated with Nissl solution (0.1% randomly selected starting point (E or W) and allowed to swim toluidine blue in walpole buffer 0.2 M and pH 4.5) at room until it escaped onto the platform, elevated 1 cm above the water temperature for 3 min and washed with distilled water. Sections level in the N position and indicated by a visible striped flag were dehydrated, cleared in xylene, and coverslipped with (5.3 × 8.3 × 15 cm). Extra maze cues were absent in the black DPX. painted walls of the room. Statistics Place Learning With a Hidden Platform Results are expressed as means ± SEM. Repeated measures On the following day, the place learning task consisted of three ANOVA (RMA) with a 2 × 2 × 2 HI × NH × S factorial analysis trial sessions per day for 4 days with trials spaced 30 min with HI, NH and S (sex) as main factors and within subjects A apart. The mouse was gently released (facing the wall) from one (Age) or T (Time course) analysis was used followed by post hoc randomly selected starting point (E or W, as these are equidistant tests. Two independent measures were analyzed with Student’s from the target) and allowed to swim until escaped onto the t-test, while those obtained for the same animals at PND23 and Frontiers in Behavioral Neuroscience | www.frontiersin.org 5 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury PND70 were analyzed with paired t-test. Behavioral correlations by the injury scores and histological appearance (only males with brain damage were analyzed with Pearson’s correlation. shown). HI animals presented high injury scores as compared P < 0.05 was considered statistically significant. to null values in the two control groups, sham and handled. HI + NH animals were not exempt of injury (vs. sham or handling, all F (1,65) > 7.9463, P < 0.009), but the total RESULTS score and that at the different neuroanatomical areas was significantly decreased as compared to HI animals. When we Short and Long Effects of HI and Their measured the injury scores in the different brain regions, Counteraction by Handling we observed that HI + NH males were different from Neuropathological Analysis HI in most regions; however, in females, the significant HI and NH effects and HI × NH interaction were found differences only appeared when we measured neocortex in the neuropathological analysis (Figure 2). No sex effects and the corpus callosum (CC) atrophy (Student’s t-test, all were detected at the level of brain injury induced, as shown P < 0.04). FIGURE 2 | Neuropathological analysis after hypoxic/ischemic (HI). (A) Drawing modified in Adobe Photoshop CS showing brain areas analyzed for quantification of brain damage in the ipsilateral side. CX, cortex; CC, corpus callosum; H, hippocampus. (B) Nissl staining showing HI effects on the cortex, hippocampus and CC of the contralateral (right side of the panel) and ipsilateral (left side of the panel) hemisphere 90 days after hypoxia. (C) Graphs show the changes in the total injury score along with the injury score in different regions analyzed. HI males and females presented higher injury scores than sham and HI + NH mice when we analyzed total injury score, the last ones were also different from sham. When we measured the injury scores in the different brain regions, we observed that HI + NH males were different from HI in most regions; however, in females, the significant differences only appeared when we measured the CC atrophy. Results are presented as mean ± SEM. Statistics: repeated measures analyses of variance (RMA), two-way ANOVA: “HI” Hypoxia effect; “NH” handling effect; “S” sex effect; “A” age effect ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. Student t-test. ∗ P < 0.05 vs. sham of the same sex; # P < 0.05 vs. handling of the same sex; § P < 0.05 vs. hypoxia of the same sex. Frontiers in Behavioral Neuroscience | www.frontiersin.org 6 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury Somatic Development (Primary Screening) responses and HI × NH interaction effects were observed in the coordination and equilibrium. However prehensility (elements Body Weight (BW) PND23–PND70–PND90 of supports when holding from a hanger) was not improved by HI resulted in slower weight gain (Figure 3A) that was handling. sex-dependent and worsened with age (both P < 0.001). Less weight gain was apparent in males since PND70, while in female Cylinder Test (CYT)—PND23 the differences were observed later, at PND90 (S × A, P < 0.001). In the CYT (Figure 4) HI increased the incidence of unimpaired Handling was not able to reverse this HI effect. paw preference in both males and females, (P < 0.001) and handling was able to reserve this effect, but only in males (P < 0.05). However, the detailed analysis of the use of the Sensorimotor Functions and Motor Activity paws (used alone, alternatively, both at a time and the total (Secondary Screening) number of vertical rearings) indicated that NH exerted beneficial Sensorimotor Functions (SMT)-PND23-PND70 effects also in females, as they showed an increased number At the sensorimotor level (Figure 3B), HI females showed of rearings using the impaired right arm, and were able to impairment of visual and hind limb extension reflexes, even alternate both arms (F’s(1,65) > 3.168, P < 0.05). Latency of at PND23 and worsening at PND70, which was the same for first self-grooming behavior showed a clear sex-dependent effect, males of this age (all, P < 0.05). Prehensility, equilibrium and with females being faster in the expression of this emotivity- coordination measured in the wire rod and hanger tests were related behavior. All the other groups, showed fast elicitation also found to be impaired and were in this order of severity of it, too. However, in the male sex, the total number of (P < 0.001, P < 0.01 and P < 0.05, respectively). Handling episodes made a difference between HI-animals and those with was able to modulate the HI-induced impairment in reflex NH or HI+NH. It is also important to note the appearance of FIGURE 3 | (A) Somatic development [postnatal day (PND)23–PND90] and (B) sensorimotor functions (PND23–PND70). Short- and long-effects of HI in males and females C57BL6 and effects of neonatal handling (NH). Results are presented as mean ± SEM. HI animals presented slower weight gain, with male being more sensitive than female; handling could not reverse the hypoxia’s effect. At sensorimotor level, HI impaired visual and hindlimb reflexes, which were first shown in females (PND23). Equilibrium, prehensility and coordination were also affected. Handling modulated the impaired reflex responses and an interaction with HI was also seen in other sensorimotor functions. Statistics: RMA, two-way ANOVA: “HI” Hypoxia effect; “NH” handling effect; “S” sex effect; “A” age effect ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, followed by Duncan’s post hoc test. ∗ P < 0.05 vs. sham of the same sex; # P < 0.05 vs. handling of the same sex; § P < 0.05 vs. hypoxia of the same sex; s P < 0.05 female vs. male of the same treatment. Paired t-test comparison day 23 vs. day 70, a P < 0.05. Frontiers in Behavioral Neuroscience | www.frontiersin.org 7 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury FIGURE 4 | Cylinder Test (CYT): short- and long-effects of HI in males and females C57BL6 and effects of NH. HI of both genders and HI + NH females presented higher paw preference scores (>50%) which indicate a greater reliance on the ipsilesional limb. Moreover, these groups presented a greater number of left rearings and H + NH female also presented more alternate left and right contacts. When we measured the total rearings performed, handling and HI + NH males presented the higher scores. Latency of first self-grooming behavior showed a clear sex-dependent effect, with females being faster in the expression of this emotivity-related behavior. All the other groups, showed fast elicitation of it, too. However, in the male sex, the total number of episodes made a difference between HI-animals and those with handling or HI+NH. Latency of pop-corn behavior was shorter in groups with handling, reaching statistical significance in handled males. Statistics: RMA, two-way ANOVA: “HI” Hypoxia effect; “NH” handling effect; “S” sex effect; “A” age effect ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, followed by Duncan’s post hoc test. ∗ P < 0.05 vs. sham of the same sex; # p < 0.05 vs. handling of the same sex; § P < 0.05 vs. hypoxia of the same sex; s P < 0.05 female vs. male of the same treatment. burst, attempts to initiate the self-grooming, as a characteristic (Figure 5). However, HI+NH females presented an increased of HI-males, that was restored to normal levels in HI+NH activity. Like in the CYT, NH males presented a significant (F’s(1,65) > 2.37, P < 0.05). Latency of pop-corn behavior was increase in rearings in comparison to sham, HI female and shorter in groups with NH, reaching statistical significance in NH HI+NH male (P < 0.05). males (P < 0.05). Non-Cognitive and Cognitive Functions Spontaneous Motor Activity Test (ACT)–PND23 At PND23 in the spontaneous motor ACT, the time course (Tertiary Screening)–PND70–PND90 and total motor activity counts recorded in a 30 min period Corner Test (CT) and Open Field (OF) indicate that HI increased the horizontal component of activity in Significant effects of HI, NH and S were found in the CT males (vs. sham, P < 0.05) and that NH counteracted this effect (Figure 6A). Horizontal activity measured by numbers of Frontiers in Behavioral Neuroscience | www.frontiersin.org 8 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury FIGURE 5 | Spontaneous motor ACT: short- and long-effects of HI in males and females C57BL6 and effects of NH. Motor activity (PND23). Horizontal activity in ACT was increased in HI males and HI + NH females; however, the vertical activity was higher in handling males. Statistics: RMA, two-way ANOVA: “HI” Hypoxia effect; “NH” handling effect; “S” sex effect; “A” age effect ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, followed by Duncan’s post hoc test. ∗ P < 0.05 vs. sham of the same sex; # P < 0.05 vs. handling of the same sex; § P < 0.05 vs. hypoxia of the same sex; s P < 0.05 female vs. male of the same treatment. corners visited was reduced in HI and HI + NH mice. Vertical No differences in self-grooming or defecation were recorded activity was also influenced by hypoxia and handling, with (Figure 6B). these animals showing higher latencies to perform a first rearing and a reduction in the total number of rearings (all Dark-Light Box Test (DLB) F’s(1,65) > 5.457, P < 0.05). HI males were more neophobic HI effect was observed when we measured the number of than sham and HI females; NH and HI+NH animals of entrances into the lit area (Figure 6C), where HI males and both genders were also more neophobic than their respective HI+NH females exhibited reduced number of entries. Stretch shams. attendance activity reflected HIxS and HIxNHxS interactions (all In the OF Test, the ethogram (sequence of behavioral events) F’s(1,65) > 4.154, P < 0.05). HI males also presented a reduced was faster in sham, handled males and females and only in number of stretch attendance in comparison with sham and HI HI and HI+NH females reaching the statistical significance in females. No more significant anxiety changes were detected in most of the latencies studied (all P < 0.05). Accordingly, the the other variables. 1st minute of test was the most sensitive showing HI, NH and G differences with HI × NH, HI × S and NH × S T-Maze Test (TM) interactions (F’s(1,65) > 5.038, P < 0.05), especially in vertical No changes in working memory were apparent in the TM. activity. Regarding the locomotor activity, HI effects with Differences in the first latency to reach the intersection NH × S and HI × NH × S interactions were showed (Figure 6D) were found with significant HI effect and NHxS when we study the total vertical activity (F’s(1,65) > 7.401, interactions (all F’s(1,65) = 5.326, P < 0.05). The highest latency to P < 0.05). HI males performed less rearings than sham, reach the intersection point of the TM was observed in HI+NH HI+NH males and HI females while HI+NH females performed females as compared to other counterparts. Moreover, HI males lesser rearings than sham, HI and NH females (P < 0.05). presented a higher first T-latency in comparison with sham, Moreover, in total horizontal activity, HI males performed although there is no significant difference. HI male also spent lower number of crossing than HI females (P < 0.05) more time reaching the intersection. Frontiers in Behavioral Neuroscience | www.frontiersin.org 9 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury FIGURE 6 | Corner test (CT). OF. DLB. TM: short- and long-effects of HI in males and females C57BL6 and effects of NH. Non-cognitive functions were evaluated during PND70–PND90. Results are presented as mean ± SEM. (A) The neophobia in the CT was increased in hypoxia males and in both handling and HI + NH males and females. HI induced anxiety-like behavior in the OF (B) that lead to reduced exploratory activity with males being mostly affected. The anxious profile could be also observed in several variables measured by DL (C) were HI males and HI + NH females performed fewer entrances into the lit area. Moreover, HI males presented also fewer number of stretch attendance. Finally, in the TM (D) the highest values in the first T-latency were spent by HI + NH female, although there’s no significant difference, HI male also spent more time to reach the intersection. Statistics: RMA, two-way ANOVA: “HI” Hypoxia effect; “NH” handling effect; “S” sex effect; “A” age effect ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, followed by Duncan’s post hoc test. ∗ P < 0.05 vs. sham of the same sex; # P < 0.05 vs. handling of the same sex; § P < 0.05 vs. hypoxia of the same sex; s P < 0.05 female vs. male of the same treatment. Morris Water Maze (MWM) task learning, when the cue was removed and the platform was Figure 7A illustrates the ‘‘day-by-day’’ (left panel) and ‘‘trial- hidden, animals exhibited different acquisition curve. The HI by-trial’’ (right panel) acquisition curves. All days presented the and HI + NH animals found the hidden platform slower along temporal effect (all F’s(1,65) = 9.319, P < 0.001), especially in place the 4 days of the test as showed by a longer distance covered Frontiers in Behavioral Neuroscience | www.frontiersin.org 10 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury FIGURE 7 | MWM: short- and long-effects of HI in males and females C57BL/6 and effects of NH. Cognitive functions were evaluated during PND70–PND90. Results are presented as mean ± SEM. Mean distance to reach the platform in the cue and place tasks for spatial learning. All days presented time effect. (A) Cognitive deficits were observed in the MWM in both sexes with reduced total learning capacities (acquisition of the task) and worse short and long-term learning. In HI mice a reduction of the mean distance covered to find the platform could be recorded in both “trial-by-trial” and “day-by-day”, especially in place tasks learning and were mainly important in HI males. Memory in HI males was more clearly affected than females in the MWM, however it was modulated by handling (B) In the probe trial for short-term memory, hypoxia males did not distinguish between the trained quadrant (P) and the adjacent (Ar, Al) or the opposed one (O) presenting a random swimming while handling (male and female) and HI + NH male presented a scanning swimming. Finally sham (male and female), HI and HI + NH female presented a focal searching swimming due they distinguish between the trained quadrant and the rest of the quadrants. The number of annulus crossings was also fewer in HI and HI + NH males. Statistics: Duncan’s post hoc test. ∗ P < 0.05 vs. sham of the same sex; # P < 0.05 vs. handling of the same sex; § P < 0.05 vs. hypoxia of the same sex; s P < 0.05 female vs. male of the same treatment. Preference for the trained quadrant (P) as compared to adjacent right (Ar) and left (Al) or Opposite (O) quadrants in the probe trial. ANOVA, “Q” preference for trained quadrant. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001; followed by Duncan’s post hoc test ∗ P < 0.05 vs. P quadrant, o P < 0.05 vs. O quadrant. n.s. non-significant statistical differencs is found. to find the platform in comparison to sham mice (HI and S opposed ones (O) presenting a random swimming. In contrast effects; HI × NH, HI × S and HI × NH × S interactions, handled (male and females) and HI+NH males presented a F (1,65) > 3.908, P < 0.05). Memory in HI males was more scanning swimming. Finally, sham (male and female), HI and clearly affected than females in the probe trial of the MWM HI+NH females presented a focal searching swimming as they (Figure 7B) for short-term memory. HI males did not distinguish distinguished between the trained quadrant and the rest of the between the trained quadrant (P) and the adjacent (Ar, Al) or the quadrants (Lang et al., 2003; all P < 0.05). Although we observed Frontiers in Behavioral Neuroscience | www.frontiersin.org 11 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury FIGURE 8 | Neuroanatomical correlates of short- and long-term behavioral effects of HI in males and females C57BL/6 and those of NH. The graphical abstract summarizes the strongest neuroanatomical-behavioral correlations (P < 0.01) found in males (55 variables) and females (17 variables). This distribution, indicates that in spite of similar injury score, the NH was more effective restoring functions in males and that they were those related to the cognitive domains. In contrast, the domain of non-cognitive function was the more benefited in HI + NH females. FIGURE 9 | Graphical abstract of sex- age- and task-dependent behavior impact of HI brain injury and its modulation by handling. that this memory effect in males was modulated by handling, HI Figure 8) between those variables where HI showed a deleterious and HI + NH males presented a worse performance when we effect and/or handling an effect (see Figure 9). Neonatal handled analyzed the annulus crossing in comparison to sham (HI effect and sham animals were excluded from this analysis, since F (1,65) = 17.047, P < 0.001, post hoc Duncan’s test, all P < 0.05). injury score was null. As detailed in Figure 8, the strongest correlations (P < 0.01) were found in 55 cases in male variables Correlations Analysis as compared to 17 cases in females. This distribution indicates In a matrix of 175 × 9 (behavioral × injuries score) variables that in spite of a similar injury score, the NH was more studied, we performed a meaningful correlation analysis (see effective in restoring functions in males and within those Frontiers in Behavioral Neuroscience | www.frontiersin.org 12 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury related to the cognitive domains. In contrast, the domain In contrast to other early life interventions studied in the of non-cognitive function was improved more in HI + NH literature, we unveil for the first time a relevant sex-dependent females. improvement of NH on HI, with HI + NH males being more responsive than females. This is important to note DISCUSSION since sex differences have been considered in recent years. It has been reported that male infants are more vulnerable to The present work addresses the short and long-term behavioral perinatal insult than female infants, and they also suffer more and functional impact of neonatal HI brain injury in a term long-term cognitive deficits. Males showed increased risk of (PND7) mice model using a longitudinal approach and development disorders, including speech and language, autism, taking into consideration sexual dimorphism. It also provides learning disabilities and cerebral palsy compared to females evidence of a remarkable neuropathological protection elicited (Donders and Hoffman, 2002; Rutter et al., 2003; Marlow by NH. This tactile and proprioceptive sensory stimulation et al., 2005; Tioseco et al., 2006; Hill and Fitch, 2012). Some was administered to mice from PND1, a temporal frame authors also refer that being ‘‘male’’ has been identified as a modeling premature children, when the human immature universal risk factor for the incidence of neonatal stroke as brain is at increased risk of HI injury, mostly in males. well as developmental delays (Elsmén et al., 2004; Peacock NH not only ameliorated the behavioral outcomes and et al., 2012; Månsson et al., 2015). Moreover, after pediatric functional capacities but also showed differences in a sex- traumatic brain injury, girls demonstrate a significantly better age- and task-selective manner. Several levels of study were outcome in tests of learning and memory (Donders and considered from behavior to neuropathology, including key Hoffman, 2002; Hurn et al., 2005). In our present work, HI areas of clinical interest such as BW, sensorimotor function, males showed a worse performance in the memory test as physical/motor activity, emotionality, cognitive function compared to HI females and the improvement induced by NH and finally brain pathology in different neuroanatomical in HI males was supported by better levels of hippocampal regions. The evaluation of these risk/protective events studied preservation. at weaning and adulthood indicates that benefits observed Behavioral and functional phenotype assessment was in the developmental outcome also result in long-term performed using a three-stage protocol (Giménez-Llort et al., sustained effects, which contributes to their translational 2002). The somatic development was evaluated as primary interest. Besides, the results indicate that even with similar screening, and BW was used for that. Feeding dysfunction and HI-induced brain injury scores, interaction effects with nutritional problems has been associated with poor growth and sex are important to be taken into consideration when health status in children with cerebral palsy and neurological assessing the outcome of preventive and/or therapeutic impairment (i.e., Reilly et al., 1996; Sullivan et al., 2000; strategies. Fung et al., 2002). Experimentally, the few number of studies In contrast to most studies describing detailed addressing this issue has shown lower daily weights in HI rats neuropathological changes in the rat model, here we provide (Andiné et al., 1990; Balduini et al., 2001; Lubics et al., 2005; the neuropathological tissue damage at 90 days, and the Girard et al., 2012). Here we found differences in growth rate neuroanatomical distribution in both male and female of HI mice that worsened with age and were long-lasting. The C57BL/6 mice strains. In agreement with a devastating results also indicate that in males sex BW was more sensitive to early PBI insult, the histopathological analysis of HI brains the impact of HI than in females, who exhibited lower weight provided evidence of the severity of the experimentally later in adulthood. This was in agreement with the sex-dependent induced brain injury, and the histopathological protection vulnerability that we found in the behavioral outcome. conferred by NH intervention. PBI is considered as one of The secondary screening evaluated the sensorimotor the major contributors to perinatal morbidity and mortality. functions and motor activity. Strength, motor coordination Here, the mean index of postnatal mouse mortality due to and several reflex responses (righting, geotaxis, gait and cliff surgery or hypoxia was 19.31%, with 18.46% for males and aversion) were reported as impaired after HI insult in rats and 20.00% for females, showing no statistical differences between mice (Ten et al., 2003; Fan et al., 2005; Lubics et al., 2005; sexes. Karalis et al., 2011). Here, other sensorimotor tasks such as High variability in size and severity of the infarct between the visual and extension reflexes, prehensility or grasping, animals is a significant drawback of the HI experimental model equilibrium and coordination were found also impaired by HI. (Vannucci and Vannucci, 2005; Millar et al., 2017). It seems that Again, for the first time, we show the relevance of ‘‘sex and sex, severity, time of injury or even the brain lateralization of age’’ factorial interaction as well as the comparative degree the lesion can significantly affect the outcome of Rice-Vannuci of functional severity. Spontaneous functional recovery in model (Lubics et al., 2005; Sanches et al., 2013b, 2015). In this motor coordination and righting, geotaxis and gait reflexes regard, it is noticeable that, in the present work, the HI-procedure were found in some of those previous works (Lubics et al., induced homogeneous total injury scores and neuroanatomical 2005; Karalis et al., 2011). In our case, in spite of the high distribution in both sexes. Most importantly, this allowed us maturation of motor systems in adults, these tasks were more to highlight the capacity of NH to reduce the injury score in demanding for them, due to increased body size vs. the rods most of the areas, albeit not all of them reached statistical widths. This could explain that functional motor recovery was significance. only seen for HI-induced hyperactivity in males. Thus, HI-males Frontiers in Behavioral Neuroscience | www.frontiersin.org 13 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury spontaneously returned to the normal values shown by sham that although HI were more active; when locomotion requires animals, but deficient reflexes were still evident at 70 days of a higher level of coordination, mice can be hypoactive. Short age. test duration (Chou et al., 2001) and assessment in the light On the other hand, this persistence of sensorimotor period (Antier et al., 1998) can also elicit reduced activity in HI impairments until adulthood makes the beneficial effects induced rodents, probably because under these conditions they reflect an by NH more remarkable, mostly in the reflex responses. This anxiogenic response. Thus, in the present work we show that early life tactile and proprioceptive sensory stimulation reversed the expression of neophobia and anxiety-like behaviors depend not only the impairment in reflexes but also exerted beneficial on the anxiogenic conditions of the test. In mild anxiogenic effects on coordination and equilibrium. Senses of touch, conditions the animals were found hyperactive, exhibiting a balance and proprioception are the first of the seven sensory hyperexcitability stage, while with higher illumination they systems developed during ontogeny. Thus, in the present work, showed reduced exploratory activity. This was consistent with the improvement of the vestibular system can be considered the behavioral responses shown in the other tests (the DLB a notorious effect of NH, as compared to sensory outputs and the performance in the long arm of the TM) and is achieved in other studies (Paolucci et al., 2015). It is likely also in agreement with other works (Girard et al., 2009, 2012; that the handling protocol, that involves the animals being Carletti et al., 2012; Sanches et al., 2013a,b; Soares et al., held by the experimenter and the pups individually resting 2013). in the cage, may constitute a scenario where the vestibular Academic performance and intellectual abilities are important system is challenged, trained and reinforced. In fact, early aspects in children with neonatal encephalopathy (Robertson and tactile and vestibular stimulations were postulated as crucial Finer, 1988, 1993; Moster et al., 2002; van Handel et al., 2007). for motor behavior development (i.e., Labarba et al., 1974; At a translational level, cognitive impairment has been reported Clark et al., 1977) with the maturation rate of inhibitory many times, especially in the MWM. Learning impairments systems (Oakley and Plotkin, 1975) as a hypothesis to explain (Young et al., 1986; Ikeda et al., 2001; Ten et al., 2003; de Paula hyperexcitability stages, such as the pop-corn behavior shown et al., 2009; Arteni et al., 2010) related to a longer time to escape by mice (Wahlsten, 1974) and as also observed here, in terms of in ischemic group (Balduini et al., 2001; Chou et al., 2001; Ten emotional behaviors, which can be modulated by sensorimotor et al., 2004; Ikeda et al., 2006; Huang et al., 2009) and memory training (Caston and Lateurte, 1997). In agreement with the dysfunction in the probe trial (Ten et al., 2003, 2004; Huang et al., pyramid of learning postulated by Taylor and Trott, 1991 (as 2009). Like us, no impairments in swimming ability or speed cited in Williams and Shellenberger, 1994), an improvement have been observed in injured animals (Ikeda et al., 2001, 2006; in the sensory integration dysfunction induced by HI, mostly Arteni et al., 2003). Arteni et al. (2010) described lateralized and in males, should also facilitate the improvement in learning sex-dependent behavioral and morphological effects of unilateral capacities, as shown by our results. In the histological analysis, neonatal cerebral HI in the rat. In other works (Ikeda et al., 2001; the injury score of the underlying neuroanatomical areas was Arteni et al., 2003) only animals that suffered a right HI injury reduced, but further experiments with detailed evaluation of performed worse in the working memory tasks. This lateralized these areas will provide clues about this sensory integration effect could explain why, in our case, working memory is not hypothesis. affected in HI animals. Regarding the motor development, although some authors To the best of our knowledge, there are no reports have described no impairment in the CYT (Sanches et al., 2013a), regarding sex differences in the functional recovery following most of them reported that HI causes a preference to use the HI in neonatal handled animals. Nesting environment (Mason unimpaired forepaw (Grow et al., 2003; Chang et al., 2005; Jones et al., 2018), rehabilitative training (Tsuji et al., 2010) et al., 2008; Kim et al., 2008; Lee et al., 2010; van Velthoven or early-life interventions based on environmental rearing et al., 2010; van der Kooij et al., 2010; Pazos et al., 2012), as conditions (Pereira et al., 2007, 2008; Fan et al., 2011; Rojas was also shown in the present work. In agreement with literature et al., 2013, 2015; Nie et al., 2016; Schuch et al., 2016) that (Fan et al., 2011, 2013), no differences between male and female share mechanisms of action with NH (Fernández-Teruel et al., were observed. The poorer motor skills in children with neonatal 2002), also show sex-specific neuroprotection patterns. In these encephalopathy compared to control could be related with the works, the sex differences analyzed in the recovery of HI after size of the CC (Van Kooij et al., 2008). This is important to note, environmental enrichment or rehabilitative training in rats, since in the present work the CC is the area that shows, in both described partial recovery in working memory in adolescent sexes, a statistical significant reduction of injury score in mice rats (Pereira et al., 2008) and improved swimming time and receiving NH. length in females but not in males after rehabilitative training The tertiary screening assessed locomotor activity and (Tsuji et al., 2010). Thus, it is remarkable to note that the non-cognitive functions. It is well established that hippocampal above mentioned studies showed sex-specific neuroprotection areas are highly vulnerable to HI, and that hippocampal injury patterns, but with female sex as the most resilient, while males leads to hyperactivity (Shen et al., 1991). Most of the studies seemed to be less responsive to the interventions. On the report that HI mice present hyperactivity in spontaneous ACTs other hand, different protocols for maternal separation lead to or in the OF test (Balduini et al., 2000, 2001; Ten et al., distinct behavioral outputs, from behavioral protection without 2004; McAuliffe et al., 2006; Arteni et al., 2010; Schlager et al., morphological changes (Chou et al., 2001) or reduction of 2011; Rojas et al., 2013). Lubics et al. (2005) also detected hippocampal CA volume (Lehmann et al., 2002) to worsening Frontiers in Behavioral Neuroscience | www.frontiersin.org 14 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury of the effects of neonatal HI (Tata et al., 2015). In other without disability (Tagin et al., 2012). Despite the efficiency of experimental models of aging, neurological and psychiatric hypothermia, it is not enough to prevent all injury or neurological diseases, NH has also demonstrated positive effects in behavior, symptoms. Brain damage in term newborn infants therefore such as a reduction of anxiety-like behavior or an improvement remains a clinical problem due to there being limited therapeutic in learning and memory (Levine and Otis, 1958; Alasmi et al., outcomes and since research is constrained by obvious ethical 1997; Gschanes et al., 1998; Raineki et al., 2014). Also, we have limitations. The therapeutical approaches investigating how to previously shown that NH has long-term effects on reducing prevent or minimize the consequences of the HI insult have the impact of N-Methyl-D-aspartate (NMDA) excitotoxicity, reported efficacy of handling depending on the severity of the reducing the incidence of seizures, their number and severity damage (Chou et al., 2001). In our study, we demonstrate for the in rats psychogenetically selected for high- and low-avoidance first time that, under similar injury conditions, males are the sex (Fernández-Teruel et al., 2002). Furthermore, we have proved with better responsiveness to this early life intervention, mostly that the behavioral outcome in brain damage related to at the neuropathological level, as shown by the injury scores Alzheimer’s disease can be modulated by NH, in both males and the different number of areas protected. To a lesser extent, and females at adulthood (Cañete et al., 2015) and even at very this protective effect on the neuropathological consequences of advanced stages of disease in 17-month-old triple-transgenic HI insult also has a functional impact on the behavioral output. mice (Torres-Lista and Giménez-Llort, 2015). This was shown by the better performances in some tasks and Many studies reported morphological, biophysical and the neuropathological correlates that point out distinct neuronal biochemical changes following HI brain insult, especially in substrates underlying the sex- and age- related functional ipsilateral cerebral cortex, hippocampus, striatum and thalamus, impacts of these risk/protection interventions on sensorimotor, after arterial occlusion (Towfighi et al., 1991; Huang and behavioral and cognitive outcomes from ontogeny to adulthood. Castillo, 2008 ; and our own precedent work Shrivastava In conclusion, HI brain damage affected motor development et al., 2012), but there is scarcity of data to understand and sensorimotor functions, and induced hyperactivity at the consequential behavioral changes. Similarly, although weaning; anxiety-like behaviors and cognitive deficits during morphological neuroprotective action in the hippocampus was the adulthood in a sex- and age-selective manner. At the reported after tactile stimulation (Rodrigues et al., 2004), no functional level, handling reversed the impaired reflex responses behavioral outcomes were evaluated. Therefore, in the present and allowed improvement in memory performances in the study, we also aimed to estimate the translation of the injury hippocampal-dependent spatial learning test (MWM), in males. score on function for both risk (HI) and protection (NH) At an individual level, remarkable neurological protection interventions. We looked for meaningful correlations related to elicited by NH correlated with improved functional capacities. behavioral variables, showing the functional impact of damage Strong correlations were found between the sensorimotor, due to brain injury and its protection by NH. On the one behavioral and cognitive outcomes and the injury scores based hand, the analysis of neuropathological correlates shows that on atrophy, neuronal densities and cellularity in the different the level of damage induced/restored, measured in terms of affected areas (hippocampus, caudate/putamen, thalamus, atrophy, neuronal densities or cellularity in the affected areas, neocortex and CC). These neuropathological correlates point can be functionally correlated with behavioral variables. On at distinct neuronal substrates underlying the functional the other hand, the behavioral correlates referred to changes capacity to meet task-dependent performance demands and in the five main behavioral domains of the pyramid of neuroanatomical targets for recovery. Overall, the present learning (i.e., physical/motor, sensory, behavioral, emotional results provide evidence on a therapeutical potential of early and cognitive). The analysis identified the hippocampus as life interventions based on tactile and proprioceptive sensory the most affected area, which could explain why it was stimulation in the newborns with brain injury. It supports those difficult to completely reverse all the cognitive deficits in in the literature who defend the benefits of perinatal rearing females. Caudate/putamen, thalamus and CC showed the conditions as being important to be considered as adjuvant to highest percentages of prevention that may underline the better the current treatments. Moreover, it shows a sex-specificity that behavioral outcome in tasks dependent on these areas. benefits male sex, who were more at risk and reported to be less Concerning the translation of the experimental NH responsible to most interventions. administered to mice from PND1, it could model an early tactile and proprioceptive sensory stimulation implemented ETHICS STATEMENT on preterm infants. Since the functional sensory response of preterm children is immature (Fitzgerald, 2005), the clinical All experimental procedures were approved by the Ethical benefits may apply more specifically to older preterm infants Committee of Universitat Autònoma de Barcelona (CEEAH (>30 weeks gestational age) or HI infants post hypothermia 811) in accordance with Spanish regulations and the European treatment. The neuroprotective effect of early-life stimulation Communities Council Directives (2010/63/UE). could also be important during the pregnancy or prenatal period, as considered by Netto et al. (2018) and Durán-Carabali AUTHOR CONTRIBUTIONS et al. (2017). At the clinical level, the standard of care in cases of moderate to severe HIE is therapeutic hypothermia LG-L conceived and designed the experiments. KS and MR which has been demonstrated to increase long-term survival performed the risk/protection strategies. KS performed and Frontiers in Behavioral Neuroscience | www.frontiersin.org 15 February 2019 | Volume 13 | Article 7 Muntsant et al. Handling Protects Perinatal Brain Injury analyzed the neuropathological studies. AM performed and FUNDING analyzed the behavioral studies. LG-L, KS and AM wrote the manuscript. All authors revised and approved the final version The work received support from Fundació La Marató de TV3 of the manuscript. AM and KS equally contributed to the present 2011-110531 and Universitat Autònoma de Barcelona (UAB) work. GE260408 and EME-13-140335. REFERENCES Donders, J., and Hoffman, N. M. (2002). Gender differences in learning and memory after pediatric traumatic brain injury. Neuropsychology 16, 491–499. Alasmi, M. M., Pickens, W. L., and Hoath, S. B. (1997). Effect of tactile doi: 10.1037//0894-4105.16.4.491 stimulation on serum lactate in the newborn rat. Pediatr. Res. 41, 857–861. Durán-Carabali, L. E., Arcego, D. M., Odorcyk, F. K., Reichert, L., Cordeiro, J. 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Nasal Conflict of Interest Statement: The authors declare that the research was administration of stem cells: a promising novel route to treat neonatal ischemic conducted in the absence of any commercial or financial relationships that could brain damage. Pediatr. Res. 68, 419–422. doi: 10.1203/pdr.0b013e3181f1c289 be construed as a potential conflict of interest. Vannucci, S. J., and Hagberg, H. (2004). Hypoxia-ischemia in the immature brain. J. Exp. Biol. 207, 3149–3154. doi: 10.1242/jeb.01064 Copyright © 2019 Muntsant, Shrivastava, Recasens and Giménez-Llort. This is an Vannucci, R. C., and Vannucci, S. J. (1997). A model of perinatal hypoxic-ischemic open-access article distributed under the terms of the Creative Commons Attribution brain damage. Ann. N Y Acad. Sci. 835, 234–249. doi: 10.1111/j.1749-6632.1997. License (CC BY). The use, distribution or reproduction in other forums is permitted, tb48634.x provided the original author(s) and the copyright owner(s) are credited and that the Vannucci, R. C., and Vannucci, S. J. (2005). Perinatal hypoxic-ischemic original publication in this journal is cited, in accordance with accepted academic brain damage: evolution of an animal model. Dev. Neurosci. 27, 81–86. practice. No use, distribution or reproduction is permitted which does not comply doi: 10.1159/000085978 with these terms. Frontiers in Behavioral Neuroscience | www.frontiersin.org 19 February 2019 | Volume 13 | Article 7