Therapeutic hypothermia for the treatment of neonatal hypoxia-ischemia: sex-dependent modulation of reactive astrogliosis

doi:10.21203/rs.3.rs-1333012/v1

Download PDF

Research Article

Therapeutic hypothermia for the treatment of neonatal hypoxia-ischemia: sex-dependent modulation of reactive astrogliosis

https://doi.org/10.21203/rs.3.rs-1333012/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Therapeutic hypothermia (TH) is the standard treatment for neonatal hypoxia-ischemia (HI) with a time window limited up to 6h post injury. However, influence of sexual dimorphism in the therapeutic window for TH has not yet been elucidated in animal models of HI. Therefore, the aim of this study was to investigate the most effective time window to start TH in male and female rats submitted to neonatal HI. Wistar rats (P7) were divided into the following groups: NAÏVE and SHAM (control groups), HI (submitted to HI) and TH (submitted to HI and TH; 32ºC for 5h). TH was started at 2h (TH-2h group), 4h (TH-4h group), or 6h (TH-6h group) after HI. At P14, animals were subjected to behavioural tests, volume of lesion and reactive astrogliosis assessments. Male and female rats from the TH-2h group showed reduction in the latency of behavioral tests, and decrease in volume of lesion and intensity of GFAP immunofluorescence. TH-2h females also showed reduction of degenerative cells and morphological changes in astrocytes. Interestingly, females from the TH-6h group showed an increase in volume of lesion and in number of degenerative hippocampal cells, associated with worse behavioral performance. Together, these results indicate that TH neuroprotection is time- and sex-dependent. Moreover, TH started later (6h) can worsen volume of brain lesion in females. These data indicate the need to develop specific therapeutic protocols for each sex and reinforce the importance of early onset of the hypothermic treatment.

neonatal hypoxia-ischemia

therapeutic hypothermia

GFAP

hippocampus

neurodevelopment

astrocytes

Neonatal hypoxia-ischemia (HI) is one of the main causes of mortality and morbidity in neonates (Nelson and Lynch 2004; Shankaran et al. 2005). Moreover, HI survivors can develop cerebral palsy, neurosensory deficits and cognitive impairments throughout life (Shankaran et al. 2005). Currently, therapeutic hypothermia (TH) is well established as the standard treatment used for neonates with moderate to severe injury caused by neonatal HI (Abate et al. 2021). TH must be started within a period of 6 hours after the hypoxic event (therapeutic window) in order to achieve desired neuroprotective effects (Gunn et al. 1997; Cho et al. 2020). In humans, the recommended protocol for TH is reduction of the temperature of the newborn to 33–34ºC for 72 hours (Davies et al. 2019). However, in experimental settings, the efficacy of hypothermia protocols seems to depend on the species studied (Gunn et al. 1997; Wood et al. 2016). A hypothermia of 30ºC lasting 120h showed the best neuroprotection in sheep when started 5.5h after hypoxic-ischemic insult (Gunn et al. 1997). However, in rats, TH protocols using longer periods and lower temperatures do not show additional neuroprotection and can even increase brain injury (Sabir et al. 2012, 2014; Wood et al. 2016).

TH is used in clinics irrespective of patients’ sex (Shankaran et al. 2005; Cho et al. 2020). However, there is growing evidence of the existence of sexual dimorphism in neural responses following HI, especially in animal models (Netto et al. 2017); thus, it is conceivable that neuroprotective effects of TH could be influenced by sexual dimorphism. In fact, some studies in animal models of HI have shown that neuroprotective effects of TH may depend on animals’ sex (Thoresen et al. 2009; Burnsed et al. 2015; Smith et al. 2015, 2016; Nie et al. 2016). A better perfomance in behavioural tests was observed in females treated with TH as compared to males, whereas other studies found no sex-related differences on the volume of injury or the behavioural responses when sex was considered as a variable (Wagner et al. 2002; Sabir et al. 2012, 2014; Fang et al. 2013).

Together with microglia activation and peripheral leukocyte infiltration into the nervous tissue, reactive astrogliosis plays a key role in the extension of the injury following HI. Astrocyte hyperactivity is associated with secondary neuronal damage due to release of proinflammatory cytokines after HI (Ahn et al. 2018; Escartin et al. 2021). TH is able to reduce reactive astrogliosis (Sabir et al. 2016; Ahn et al. 2018); however, it is not clear whether this reduction is influenced by sexual dimorphism.

Clinical studies demonstrate that the therapeutic window for hypothermia in neonatalogy has been set to a maximum of 6 hours after the hypoxic-ischemic event (Gunn et al. 1997; Davies et al. 2019; Catherine et al. 2021). However, there is no such definition in animal models of HI, with experimental hypothermia being applied during hypoxia or up to 12 hours post-insult (Sabir et al. 2012; Burnsed et al. 2015; Smith et al. 2015; Reinboth et al. 2016). In order to better understand the mechanisms through which TH produces its beneficial effects, an experimental time-window shall be established.

In the present study, we aimed to characterize the effective time window of TH in male and female rats submitted to neonatal hypoxia-ischemia evaluating developmental (body weight, negative geotaxis and righting reflex), morphological (brain lesion volume and degenerating cells in the hippocampus) and structural (GFAP immunoreactivity and astrocyte morphology) parameters following HI. The working hypothesis is that the effects of therapeutic hypothermia are influenced by sexual dimorphism.

2.1 Animals

In this study, 208 seven-day-old (P7) Wistar rats (male and female) and 26 dams obtained from the Animal Facility of the Department of Biochemistry of the Federal University of Rio Grande do Sul, Porto Alegre, RS were used. The pups were kept in 270 x 260 x 310 mm plastic boxes with their respective mothers (08 pups per box). All animals were kept in a 12h/12h light/dark cycle with controlled temperature (± 22ºC) and mothers received food and water ad libitum. This study was approved by the Institutional Animal Care and Use Committee of the Federal University of Rio Grande do Sul (#31442).

2.2 Neonatal hypoxia-ischemia

Neonatal HI procedure was based on the Rice-Vannuci model (Rice et al., 1981) and our previous studies (Sanches et al. 2013; Fabres et al. 2018, 2020; Tassinari et al. 2020). At P7, under isoflurane anaesthesia (5% for induction and 3% for maintenance), neonates underwent surgical occlusion of the right common carotid artery. A longitudinal incision was made in the neck to allow access to the right common carotid artery which was isolated and permanently occluded using surgical thread (silk 4.0). After surgery, pups were left with their mothers for 1h-2h for recovery and then placed in a hypoxia chamber (n = 6 animals per chamber) and exposed to an atmosphere of a certified mixture of 8% O₂ and 92% N₂ for 90 minutes at 37°C. After hypoxic exposure, pups were removed from the chamber and kept in a warm box, for approximately 15 minutes, and then returned to their mothers.

2.3 Therapeutic Hypothermia

Animals were placed in an acrylic chamber inside a temperature-controlled water bath for the hypothermia procedure (water temperature: 18–20ºC). The animals reached a body temperature of 32ºC (standard temperature indicated for therapeutic hypothermia in newborn animals) (Wood et al. 2016) within 20 minutes. Animals that failed to reach this temperature within the initial 20 minutes were excluded from the experiment. Animals were maintained in the hypothermic chamber for 5 uninterrupted hours. After hypothermia, animals were rewarmed for 30 minutes until they reached a body temperature of 36–37ºC and then returned to their mothers (Wood et al. 2016).

For this study three different therapeutic time windows to start HT after HI were used: hypothermia started two hours (TH-2h group), four hours (TH-4h) or six hours (TH-6h) after hypoxic-ischemic event. From end of hypoxia exposure until beginning of hypothermia, animals from each group were kept in their boxes with their mothers. For each time window (2, 4 or 6h to start hypothermia) there were three respective control groups: NAÏVE group (animals not manipulated), SHAM group, and HI group (animals submitted to HI but kept in normothermia). These last two groups were used to monitor the differences caused by the time animals were kept away from their mothers. However, as no differences were observed among these control groups, they were combined into a single SHAM group. All experimental groups were additionally separated into males and females. Sixteen (16) animals were randomly allocated per group. For this study, a mortality rate of 7.69% of total animals was found (16/208). The timeline of the experiment is depicted in Fig. 1.

2.4 Body temperature and body weight

Body temperature of each animal during the experiments was measured using an infrared thermometer (Incoterm, TCl 1000). The accuracy of an infrared thermometer was compared to that of a rectal thermometer in a pilot study which showed differences lower than 0.5ºC between them (data not shown). Thus, we decided to continue experiments using only the infrared thermometer to avoid the stress caused by the rectal one (Smith et al. 2015).

Animal's body temperature was measured every 5 minutes during the first 20 minutes of hypothermia. All animals in the TH groups reached the temperature of 32 ± 0.5ºC within these first 20 minutes. After that, body temperature of animals from groups SHAM, HI and TH was measured every 30 minutes. Temperature of animals in the NAIVE group (kept in their own boxes) was not measured in order to reduce manipulation effects, as well as to reduce dam's stress. Every animal was weighed at two timepoints: before surgery for carotid occlusion (P7) and before euthanasia (P14).

2.5 Histological analysis

In order to assess the volume of lesion of the cerebral hemisphere, hippocampus and cerebral cortex, animals (P14) were deeply anesthetized with isoflurane and transcardially perfused with saline solution (0.9% NaCl) followed by 4% paraformaldehyde (PFA). Brains were removed and submerged in a solution of 4% PFA overnight, then dehydrated in an alcoholic series (80%, 90%, 96% and 100% ethanol) and cleared in xylene. Afterwards, brains were embedded in paraffin, sectioned in the coronal plane (7 µm) using a microtome (Microm HM 340E ThermoScientific) and stained with hematoxylin and eosin (HE).

The following antero-posterior coordinates were used for brain analysis: +2.52 mm to -6.84 mm relative to bregma for analysis of the cerebral hemisphere; +1.70 mm to -4.16 mm for the cerebral cortex and − 2.04 mm to -6.12 mm for the hippocampus. All coordinates were obtained according to the Paxinos and Watson rat brain atlas (Paxinos and Watson 2007) and to a previous study from our group (Fabres et al. 2020). In order to reduce possible errors due to occurrence of brain oedema, the equation described by Sun et al. (2015) was used to calculate the volume of lesion (Sun et al. 2015; Fabres et al. 2020).

The same images used to assess brain lesion were used to calculate a score indicative of lesion severity (n = 10), according to Thoresen and colleagues(Thoresen et al. 1996) by an evaluator blinded to the groups. Injury severity was divided into 9 levels varying from 0 (without injury) to 4 (maximum injury), with 0.5 intervals. The classification was performed based on the percentage of injury according to the scale: 0 (without lesion), 1 (injury equal to or less than 10%), 2 (indicative of 20–30% of lesion), 3 (injury between 40–60%) and 4 (more than 75% of injury). To calculate pathological score of the hippocampus, animals with percentage of lesion below or equal to 20% were classified with a score of 1; a score of 2 indicates 50% injury, a pathological score of 3 indicates 75% lesion; and a score of 4 indicates a value of 100% injury.

2.6 Cell counting

To estimate the number of cells in degeneration (cells showing shrinkage and deformity of the cell body or karyorrhectic and pyknotic nuclei) we used slides stained with haematoxylin and eosin as described in a previous paper (Fabres et al. 2020). CA1 subfields and hilus of the dentate gyrus of the hippocampus ipsilateral to the lesion were evaluated. Five different sections containing CA1 and hilus were evaluated per animal. The images were obtained using a microscope (Zeiss) at a magnification of × 400. Mean of number of degenerative cells divided by mean of total number of cells × 100 was used to estimate percentage of degenerative cells in each subfield.

2.7 Immunofluorescence and morphological analysis

Sections were deparaffinized using xylene (10 minutes) and rehydrated using a decreasing alcoholic series. After these procedures, staining was performed as previously reported (Fabres et al. 2020). For identification of astrocytes, a primary anti-glial fibrillary acidic protein antibody (anti-GFAP, #G9269, rabbit IgG, 1:200, Sigma-Aldrich) was used and the secondary antibody of choice was Alexa 488 anti-rabbit (1:500, Molecular Probes, Invitrogen). The slides were sealed with mounting medium containing DAPI (Merck, F6057) and coverslipped.

For analysis of fluorescence intensity (used as an indicative of reactive astrogliosis), 5 sections per animal were used (n = 5 animals per group); NAÏVE and SHAM groups were used as controls. All images were captured with high magnification (400 x) using a Nikon Eclipse E-600 microscope (Japan). A 3800 µm² area of optical interest was defined to delimit all areas of interest and value of integrated density per unit of area was obtained using ImageJ software (NIH, Bethesda, USA).

The assessment of astrocyte morphology (Fig. 2) in the CA1 area of the hippocampus (number of primary processes and length of processes relative to soma), was perfomed in 3 astrocytes per section (5 sections per animal, totalling 15 astrocytes per animal). Sholl's concentric circles method was used for this analysis: concentric circles with 2 µm intervals were drawn around each cell analyzed using Image-Pro Plus software 6.0 (Mestriner et al. 2011).

2.8 Behavioural Assessment

The test of negative geotaxis was performed according to Ahn and colleagues(Ahn et al. 2018) with minor modifications. At P14, each animal was placed individually on a platform with an inclination of 35º, the head turned towards the bottom of the platform. The latency of the animal to rotate 180º and position itself with its head facing top of the platform was registered (Ahn et al. 2018). Each animal was tested three times and the mean latency was calculated.

To test the righting reflex at P14, animals were removed from their cages and put on their back on a flat surface. The latency necessary to straighten up with all paws on the surface was recorded (Hermans et al. 1992).

2.9 Data analysis

All statistical analyses were performed using GraphPad Prism 6.0. Normality test was performed, and parametric data were analyzed by two-way analysis of variance (ANOVA) (factors: sex and group) followed by Bonferroni post-hoc test. Data were plotted as box-and-whiskers with boxes depicting median and 25th and 75th percentiles, and whiskers representing minimum and maximum values for each data set.

3.1 Body weight

At P7, there were no differences in body weight among groups before animals underwent neonatal HI and two-way ANOVA did not detect effect of group (F_5,180 = 0.07140, p = 0.9964) nor sex (F_1,180 = 0.03382, p = 0.8543). However, at the age of 14 days, there was a significant effect of both factors, group (F_5,176 = 20,56, p < 0.0001) and sex (F_1,176 = 14,65, p = 0.0002) on body weight, but no interaction was verified (F_5,176 = 0,4278, p = 0.8289). Animals submitted to HI showed significantly lower body weight as compared to control groups (p < 0.05), except for the TH-2h group (p > 0.05), in which both (males and females) had body weights similar to animals from the control groups (Fig. 3).

3.2 Brain injury volume

Figure 4A shows volume of lesion in the right cerebral hemisphere. Two-way ANOVA showed a significant effect of the group (F_5,180 =149.7, p < 0.0001), with no effect of sex (F_1,180 =1.626, p = 0.2050) on the volume of lesion. HI groups showed a volume of injury of the brain hemisphere as compared to NAÏVE and SHAM groups (p < 0.05). When analyzing males, TH-2h group was the only one to show a hemispheric volume of brain lesion smaller then compared to the HI group (p < 0.05). In females, a significantly smaller volume of hemispheric lesion was observed in the TH-2h and TH-4h groups as compared to the HI group (p < 0.05). In addition we observed that males from the TH-2h group had a larger volume of lesion in the hemisphere compared to the TH-2h female group (p < 0.05).

Two-way ANOVA detected a significant effect of group (F_5,180 = 123.9, p < 0.0001), but not sex (F_1,180 = 0.5032, p = 0.4796), on volume of hippocampal lesion (Fig. 4B). Animals from the HI group showed significant volume of lesion as compared to NAÏVE and SHAM groups (p < 0.05). The TH-2h group showed a significantly smaller lesion volume relative to the HI group for both sexes (p < 0.05).

Analysis of the volume of lesion of the cerebral cortex (Fig. 4C) showed a significant effect of both, group (F_5,180 = 110.5, p < 0.0001) and sex (F_1,180 = 5.480, p = 0.0211), as well as an interaction between the factors (F_5,108 = 2.392, p = 0.0423). All groups submitted to HI showed an increased in cortical volumes of injury relative to the NAÏVE and SHAM groups (p < 0.05). The TH-2h group showed a significantly smaller volume of lesion as compared to the HI group (p < 0.05), for both sexes. It was also observed that males from the TH-4h group showed smaller volumes of lesion in the cerebral cortex as compared to the TH-4h female group (p < 0.05).

3.3 Pathology score

Pathology scores of the cerebral hemisphere are shown in the Fig. 4D. There was a significant effect of group (F_5,180 = 455.7, p < 0.0001), and sex (F_1,180 = 5.025, p = 0.0270) and an interaction between both factors (F_5,108 = 2.506, p = 0.0346). The HI group showed an increased pathology score as compared to NAÏVE and SHAM groups (p < 0.05). Both males and females from the TH-2h group had reduced scores compared to the HI group (p < 0.05); moreover, TH-2h females showed smaller scores as compared to the TH-2h male group (p < 0.05).

Two-way ANOVA showed a significant effect of the factor group (F_5,180 = 111.2, p < 0.0001), with no effect of sex (F_1,180 = 0.01549, p = 0.5012) in pathology score of the hippocampus (Fig. 4E). HI rats showed a significantly higher pathological score relative to NAÏVE and SHAM groups (p < 0.05). The TH-2h group showed a decrease in pathological score when compared to the HI group for both sexes (p < 0.05).

When pathological score of the cerebral cortex was evaluated (Fig. 4F), two-way ANOVA showed a significant effect for both factors, group (F_5,180 = 238.8, p < 0.0001) and sex (F_1,180 = 7.032, p = 0.0092), as well as significant interaction between factors (F_5,108 = 8.369, p = 0.0220). The HI group had a greater pathology score compared to NAÏVE and SHAM groups for both sexes (p < 0.05). In males, both TH-2h and TH-4h groups showed a reduction in pathology score relative to the HI group (p < 0.05). Among females, only the TH-2h group showed a reduced score as compared to the HI group (p < 0.05). In addition, females from the TH-4h group showed a higher pathology score in the cerebral cortex compared to the TH-4h male group (p < 0.05).

3.4 Percentage of degenerative cells

The percentage of degenerative cells was analyzed in the CA1 and hilus subfields of the hippocampus ipsilateral to carotid occlusion (Fig. 5A). In the CA1 region, two-way ANOVA detected a significant effect of group (F_5,180 = 350.4, p < 0.0001) and sex (F_1,180 = 14.22, p = 0.0003), as well as an interaction between factors (F_5,108 = 8.369, p < 0.0001). The HI group showed a higher percentage of degenerative cells in relation to the NAÏVE and SHAM groups (p < 0.05). In males, the TH-2h group showed a reduced percentage of degenerative cells in CA1 as compared to the HI group (p < 0.05). In females, a reduction in the percentage of degenerative cells was observed in the CA1 area of the groups TH-2h and TH-4h, when compared to the HI group (p < 0.05). Moreover, the percentage of degenerative cells in the CA1 area of groups TH-2h and TH-4h was higher in females than in males (p < 0.05).

When percentage of degenerative cells was evaluated in the hilus of the dentate gyrus (Fig. 5B), two-way ANOVA showed a significant effect of group (F_5,180 = 350.4, p < 0.0001) and sex (F_1,180 = 14.22, p = 0.0003), with an interaction between factors (F_5,108 = 6.056, p < 0.0001). For both sexes, HI groups followed the same pattern observed in the CA1 area, showing an increase in percentage of degenerative cells as compared to the NAÏVE and SHAM groups (p < 0.05). In males, the TH-2h and TH-4h groups showed a decrease in percentage of degenerative cells in the hilus relative to the HI group (p < 0.05). Among the females, only the TH-2h group showed a reduction in percentage of degenerative cells compared to the HI group (p < 0.05). However, a sex-related difference was also observed: in the HI and TH-2h groups a smaller percentage of degenerative cells was observed in females (p < 0.05). Besides that, the TH-6h female group showed an increase in percentage of degenerative cells in comparison to the HI female group (p < 0.05).

3.5 GFAP immunoreactivity and astrocyte morphology

GFAP immunoreactivity and astrocyte morphology were studied in the CA1 subfield of the hippocampus ipsilateral to the carotid occlusion (Fig. 6). Two-way ANOVA showed a significant effect of group (F_4,40 = 19.87 p < 0.0001) and sex (F_1,40 = 6.625 p = 0.00139) on GFAP fluorescence intensity (Fig. 6A). The HI group showed an increase in GFAP fluorescence intensity as compared to the control group, for both sexes. Males from the TH-2h and TH-4h groups as well as females from TH-2h showed reduction in GFAP fluorescence relative to the HI group (p < 0.05).

In addition, two-way ANOVA showed a significant effect of the group (F_4,39 = 31.42, p < 0.0001), with no effect of sex (F_1,39 = 1.865, p = 0.1799) when the number of primary process of astrocytes was evaluated (Fig. 6B). In the analysis of the length of processes of astrocytes (Fig. 6C), two-way ANOVA detected a significant effect of group (F_4,37 = 55.28 p < 0.3598) and sex (F_1,37 = 0.8596 p = 0.3598).

Animals from both sexes showed an increase in length of the processes in the HI group as compared to the control group; however, only females from TH-2h group had reduced length of the processes compared to the HI group (p < 0.05).Male and female HI groups showed the same pattern observed in analyses of the lenght of processes: there was an increase in number of astrocyte primary processes compared to the control group (p < 0.05) and TH treatment reduced the number of processes only in the TH-2h female group as compared to the HI group (p < 0.05).

3.6 Behavioral Assessment

Negative geotaxis and righting reflex performances are shown in Fig. 7. Two-way ANOVA showed a significant effect of group (F_5,180 = 85.66 p < 0.0001) with no effect of sex (F_1,180 = 1.361, p = 0.2449) for negative geotaxis. However, there was a significant interaction between factors (F_5,108 = 5.231, p = 0.0002). The HI group from both sexes showed an increase in latency to complete the test as compared to the NAIVE groups. In males from TH-2h and TH-4h as well as in females from the TH-2h group there was a decrease in latency to complete the negative geotaxis test relative to the HI group. However, in females from the TH-6h group, an increase was observed in the latency as compared to the HI female group (Fig. 7A).

The analysis of the righting reflex showed a significant effect of group (F_5,180 = 25.55 p < 0.0001), with no effect of sex (F_1,180 = 0.2714 p = 0.6030); an interaction between the factors was also found (F_5,180 = 2.637, p = 0.0250). The results were similar to those found for the negative geotaxis test, i.e, animals from the HI group (from both sexes) showed an increase in latency to complete the righting reflex test. However, only males from the TH-2h group reduced the latency as compared to the HI group. Moreover, females from the TH-6h group showed an increase in latency to complete the test relative to the HI group (Fig. 7B).

3.7 Correlation Analysis

Pathological score is used as a tool to estimate level of brain lesion following neonatal HI (Thoresen et al. 1996; Sabir et al. 2014). In order to assess the validity of our pathological score, we checked if these results were correlated with the results from the volume of lesion. There was a positive correlation between the pathological score and volume of lesion for all structures evaluated (cerebral hemisphere, hippocampus, and cerebral cortex) for both sexes (p < 0.05; Table 1).

Table 1

Correlation between histological and behavioural data separated by animals’ sex. ^†very weak association; ^‡weak association; ^§moderate association; *strong association; and **very strong association.
Males	Volume of Lesion			Behavioral Assessment		Immunofluorescence (GFAP)
	Hemisphere	Hippocampus	Cerebral Cortex	Negative Geotaxis	Righting Reflex	Fluorescence Intensity	Primary processes	Distance from soma
Volume of Lesion	r²	r²	r²	r²	r²	r²	r²	r²
Hemisphere	-	-	-	0.4831§	0.3017‡	-	-	-
Hippocampus	-	-	-	0.3835‡	0.3243‡	0.3430‡	0.2905‡	0.5307§
Cerebral Cortex	-	-	-	0.5017§	0.3110‡	-	-	-
Degenerative Cells
CA1 of Hippocampus	-	-	-	-	-	0.6516*	0.8092**	0.7782*
Pathology Score
Hemisphere	0.9538**	-	-	0.4368§	0.3334‡	-	-	-
Hippocampus	-	0.9084**	-	0.4505§	0.2969‡	0.3247‡	0.3250‡	0.5946§
Cerebral Cortex	-	-	0.9066**	0.4361§	0.3155‡	-	-	-
Females	Volume of Lesion			Behavioral Assessment		Immunofluorescence (GFAP)
	Hemisphere	Hippocampus	Cerebral Cortex	Negative Geotaxis	Righting Reflex	Fluorescence Intensity	Primary processes	Distance from soma
Volume of Lesion	r²	r²	r²	r²	r²	r²	r²	r²
Hemisphere	-	-	-	0.4321§	0.1882†	-	-	-
Hippocampus	-	-	-	0.5500§	0.4158‡	0.4065§	0.7136*	0.6611*
Cerebral Cortex	-	-	-	0.3514‡	0.2056‡	-	-	-
Degenerative Cells
CA1 of Hippocampus	-	-	-	-	-	0.6516*	0.8092**	0.7782*
Pathology Score
Hemisphere	0.9259**	-	-	0.4269§	0.2175‡	-	-	-
Hippocampus	-	0.9237**	-	0.4505§	0.2106‡	0.5821§	0.7026*	0.6726*
Cerebral Cortex	-	-	0.9264**	0.3103‡	0.2548‡	-	-	-

A positive correlation between the latency to complete behavioral tests and the volume of lesion or pathology score was also observed for all structures evaluated (cerebral hemisphere, hippocampus and cerebral cortex) for both sexes (p < 0.05; Table 1).

The data of volume of lesion, pathological score and percentage of degenerative cells in the hippocampus showed a positive correlation with the level of reactive astrogliosis (mainly related to length of processes and number of primary processes) for both sexes (p < 0.05; Table 1).

In this study, we have shown that the effective time window to start TH is sex-dependent. TH initiated earlier, i.e., 2 hours after injury, reduced extent of lesion in the cerebral hemisphere, hippocampus, and cerebral cortex for both sexes. However, the onset of hypothermia 6 hours after hypoxia worsened the results of morphological (percentage of degenerative cells in the hippocampus) and behavioral parameters, mainly in females. The best results of TH are achieved when treatment starts immediately or shortly after the hypoxic-ischemic event (Sabir et al. 2012; Cho et al. 2020).

Body weight is a developmental parameter which can provide important information about the protective effects of hypothermia. It is well known that HI can lead to motor deficits which, in turn, lead to feeding difficulties and affect animal’s body weight gain (Sanches et al. 2013; Fabres et al. 2018). The HI group showed a reduction in body weight as compared to SHAM and NAÏVE groups when evaluated 7 days after HI (P14). However, the TH-2h group did not show this decrease, indicating a beneficial effect of hypothermia when started 2h after HI. Such important improvement of this developmental parameter was not seen when hypothermia was started later, i.e., 4h and 6h after HI. Sabir and colleagues (2012 and 2014) did not observe any difference in weight gain between HI animals and animals treated with hypothermia (32ºC for 5h) started immediately or 3, 4, 6 or 12h after injury (Sabir et al. 2012, 2014). However, in study of Sabir et al. (2014) all animals that were submitted to neonatal HI, making it difficult to compare with our work.

The lack of weight loss observed in the TH-2h group may be associated with smaller volume of brain damage and, consequently, reduction in motor impairment. Here, animals belonging to the TH-2h group showed a decrease in lesion volume in every brain structure evaluated when compared to the HI group. However, when TH was started later (TH-4h and TH-6h groups), this neuroprotective effect of TH was blunted, suggesting TH started later may not be able to protect the brain properly. In agreement with Davson (2015) the cells can recover from an insult up to 6 hours after the event and this period is known as the latent period. Within this period, deleterious mechanisms can be initiated leading to spread of brain injury and progressive cell dysfunction (Davidson et al. 2015; Cho et al. 2020).

There are experimental and clinical evidences showing that the closer the onset of hypothermia is to the moment of HI, the greater the reduction of brain injury (Sabir et al. 2012, 2014; Thoresen et al. 2013). Sabir et al. (2012) observed that hypothermia started immediately or 3h after HI caused a reduction in brain injury, an effect that was not seen when hypothermia was started later (6h and 12h following HI) (Sabir et al. 2012). In addition, Park and colleagues (2015) did not observe neuroprotective effects when hypothermia was started 6 hours after injury (Park et al. 2015), corroborating with our findings.

In order to strengthen analysis of brain injury, we decided to calculate the pathological score(Thoresen et al. 1996) of brain structures from which the injury volume was assessed. The correlation between data of pathological score and lesion volume showed a very strong association proving both forms of analysis can be used. Although analysis of volume of brain injury and pathological score demonstrated a beneficial effect of hypothermia initiated 2h after HI, no sex-related differences were observed in such parameters. However, it is known that there is a difference in type of cell death depending on sex; in females, cell death is mainly dependent on caspase activation, whereas in males, cell death is mostly caspase-independent (Joly et al. 2004; Askalan et al. 2015; Netto et al. 2017). Therefore, an evaluation at the cellular level could help us find differences between sexes. The hippocampus was chosen for this analysis, since it is the most affected brain structure in the neonatal HI animal model (Dhikav and Anand 2012).

When percentage of degenerative cells in pyramidal layer of CA1 and hilus was evaluated, a sex-related effect was revealed. Animals from the HI group showed an 8-fold increase in percentage of degenerative cells for both sexes, which is in accordance to our previous study using only males (Fabres et al. 2020). TH reduced percentage of cells in degeneration when started up to 4h after injury, although the most remarkable neuroprotection was observed in the TH-2h group. In addition, in both hilus and CA1, the percentage of degenerative cells in females from the TH-2h group was around 15%, while in the TH-2h males this percentage was around 30% in hilus and 35% in CA1. These results corroborate with data showing that TH is able to reduce neuronal death in the CA1 area of the hippocampus three and seven days after injury (Xiong et al. 2009; Wood et al. 2016). The hilus of the dentate gyrus was chosen as being a region considered less vulnerable to HI than the CA1 area. Nevertheless, it was also shown that hypothermia started earlier was able to reduce cell degeneration in the hilus; on the other hand, when started 6h after HI (TH-6h group), the number of degenerating cells in females was even greater than that observed in the HI group.

Reduction in volume of brain injury and in percentage of degenerative cells are important factors indicative of the efficacy of TH; however, it is well known that brain injury caused by HI can extend for weeks to months (Davidson et al. 2015). Neuroinflammation is one of the main causes that extends the injury for longer periods. It is also known that male animals submitted to neonatal HI show increased microglial activation and upregulation of the inflammatory response (Netto et al. 2017). Astrocytes play a role in this process and reactive astrogliosis is also related to neuroinflammation (Davidson et al. 2015). In our study, TH started earlier (TH-2h) decreased GFAP fluorescence intensity in the CA1 area, suggesting a reduction in reactive astrogliosis for both, males and females.

However, to obtain a more detailed assessment of astrocyte activation, we used Sholl’s concentric circles method to quantify astrocyte complexity (arbour length and number of processes) (Mestriner et al. 2011; Mari et al. 2019). These parameters were evaluated because increase in GFAP immunoreactivity as well as increase in number and compliance of processes are characteristics of reactive astrocytes as already demonstrated in other studies (Haim et al. 2015; Liddelow et al. 2017; de Fraga et al. 2021). Sholl analyses showed that the TH-2h female group had a reduced number of primary processes as well as length of processes compared to the HI group, which was not observed in males. Morphology of astrocytes may reflect the functional significance of neuroglial interactions (Sheikhbahaei et al. 2018). The glial scar formation triggered by neural lesions such as those produced after hypoxic-ischemic events, when uncontrolled, may impair neural communication and cell function (Sofroniew 2015; Sheikhbahaei et al. 2018). Therefore, although GFAP fluorescence intensity was used to infer increased astrocyte reactivity, females from the TH-2h group showed smaller number of primary processes and of shorter length, indicating more organized glial scar formation; this is conceivably beneficial for tissue repair (Sizonenko et al. 2008).

Astrocyte reactivity parameters (evaluated by GFAP immunofluorescence intensity and Sholl circles) also showed a strong association with percentage of degenerative cells in CA1. It was expected, since reactive astrogliosis is a phenomenon in which astrocytes branch off and extend their processes to form a glial scar at the locations where neurons have died. A study by Reddy and colleagues (2020) also observed a correlation between volume of hippocampus and the reactive astrogliosis marker GFAP+, corroborating the hypothesis of glial scar formation (Reddy et al. 2020).

In addition to the morphological parameters, an analysis of neurodevelopmental parameters is also important and can provide broader data on the neuroprotective effects of TH. Previous studies from our group(Tassinari et al. 2020) and from others(Lubics et al. 2005) have shown that HI causes an increase in latency to complete the negative geotaxis test, as seen here. However, together with the neuroprotective effects of TH on morphological and structural variables, we have also observed an improvement in the behavioral response in the test of negative geotaxis in animals from the TH-2h group, for both sexes. Jatana and colleagues (2006) showed that hypothermia was not able to reduce the latency in the negative geotaxis test 7 days after injury; however, the hypothermia protocol lasted for only 2h, which could explain differences in the results (Jatana et al. 2006). By starting hypothermia 3 hours after injury and using a prolonged period of hypothermia (48h), Ahn and colleagues (2018) observed a reduction in latency to perform the negative geotaxis test when animals reached 42 days of life (Ahn et al. 2018), but not at P14, P21, P28, and P35. It has already been observed in animal models that a protocol of hypothermia longer than 5h produces no additional neuroprotective effects (Sabir et al. 2012). Moreover, prolonged periods of maternal separation produce negative effects on behavior (Lehmann et al. 1999), which could explain the reason why longer periods of hypothermia do not lead to improvement in behavioral outcomes.

When the righting reflex was evaluated, only males from the TH-2h group showed a reduction in latency to complete the test as compared to the HI group. Yuan et al. (2015) also observed that hypothermia tended to reduce the latency to complete the righting reflex test compared to the HI group; however, sex-dependent effects were not assessed (Yuan et al. 2015). In the present study, TH was not able to improve the female response in the righting reflex test, regardless of the period in which it was started (2h, 4h or 6h after HI). Behavioral improvements observed in males and females in the negative geotaxis test, as well as in males in the righting reflex test, may be related to the reduction in volume of lesion and decreased percentage of degenerative cells observed in these groups. To test this hypothesis, a correlation between behavioral performance, morphological and structural parameters was run. We observed only a weak to moderate association beteween these variables, depending on the parameters evaluated.

The hippocampus is a structure that play important roles in spatial navigation and episodic memory (Soltesz and Losonczy 2018). Studies using gamma radiation showed alterations in hippocampal layers with pyramidal neurons exhibiting degeneration, i.e., showing characteristics of pyknosis, karyorrhexis and karyolysis (Li et al. 2014; Owoeye and Malomo 2015). Death of pyramidal neurons can disrupt the flow of information suggesting that memory and other functions of the hippocampus could potentially be affected (Owoeye and Malomo 2015).

Furthermore, among the structures evaluated in the present study, the hilus has a regulatory role for neuronal migration in neonates, which can continue into the adult stage (Saegusa et al. 2010, 2012). Lesions in this area may suggest alterations in development and plasticity of the hippocampus, which may reflect behavioral changes. However, behavioral tests used here are characterized by reflex movements and are more related to brain areas not evaluated in this study such as the brainstem (Heyser, 2003; Schneider and Przewłocki, 2005).

Our study has some limitations. First, we evaluated animals using only one time point (P14, i.e, 7 days after the HI event) and it is well known that the brain lesion can mature from days to months (Davidson et al. 2015). Therefore, further studies using animals from both sexes treated with TH and tracked for longer periods are needed, which would allow assessment of cognitive function using specific behavioral tests in adulthood. Second, we did not evaluate molecular mechanisms underlying the neuroprotective effects of TH. It is well described that TH has an effect in reducing brain metabolism leading to delayed cellular depolarization and, thus, reduction of intracellular calcium influx; however, understanding the molecular basis of TH effects can increase knowledge of its functioning and allow the combination of TH with other adjuvant therapies, in order to increase neuroprotective benefits (Davidson et al. 2015; Cho et al. 2020).

Summarizing, we have shown that TH started early after HI injury leaded to reduction in brain damage and reactive astrogliosis, as well as an improvement in the neurological reflexes deficits caused by HI, for both sexes. However, when TH was initiated later (i.e., 6h after HI), females showed an increase in percentage of degenerative cells in the hippocampus ipsilateral to the lesion. Females from the TH-6h group also showed an increase in latency to perform behavioral tests.

To the best of our knowledge, this is the first report showing that the effects of TH on reactive astrogliosis are dependent on animals’ sex. Therefore, we emphasize the importance of starting TH as soon as possible for both sexes. If there is a need for the treatment to be started later, even greater care should be given to females, as a later start of TH treatment can intensify brain injury in females.

Funding:

This study was supported in part by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and, Financiadora de Estudos e Projetos (FINEP). We would also like to thank the Laboratório de Análises de Amostras Biológicas por Fluorescência (LabFluor - UFRGS) for the support in all analysis.

Competing interests:

The authors declare that they have no conflict of interest.

Data availability statement:

“The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request”.

Ethics approval:

All procedures involving animals were in accordance with the precepts of the National and International Guidelines, especially Law 11,794 of November 8, 2008, Decree 6899 of July 15, 2009, and as edited by the National Council for the Control of Animal Experimentation (CONCEA), was approved by the Institutional Animal Care and Use Committee of Federal University of Rio Grande do Sul (UFRGS, #31442).

Abate BB, Bimerew M, Gebremichae B, et al (2021) Effects of therapeutic hypothermia on death among asphyxiated neonates with hypoxicischemic encephalopathy: A systematic review and meta-analysis of randomized control trials. PLoS ONE 16:1–20. https://doi.org/10.1371/journal.pone.0247229
Ahn SY, Chang YS, Sung DK, et al (2018) Hypothermia broadens the therapeutic time window of mesenchymal stem cell transplantation for severe neonatal hypoxic ischemic encephalopathy. Scientific Reports 8:1–10. https://doi.org/10.1038/s41598-018-25902-x
Askalan R, Gabarin N, Armstrong EA, et al (2015) Mechanisms of neurodegeneration after severe hypoxic-ischemic injury in the neonatal rat brain. Brain Research 1629:94–103. https://doi.org/10.1016/j.brainres.2015.10.020
Burnsed JC, Chavez-Valdez R, Hossain MS, et al (2015) Hypoxia-ischemia and therapeutic hypothermia in the neonatal mouse brain - A longitudinal study. PLoS ONE 10:1–20. https://doi.org/10.1371/journal.pone.0118889
Catherine RC, Ballambattu VB, Adhisivam B, et al (2021) Effect of Therapeutic Hypothermia on the Outcome in Term Neonates with Hypoxic Ischemic Encephalopathy—A Randomized Controlled Trial. Journal of Tropical Pediatrics 67:1–6. https://doi.org/10.1093/tropej/fmaa073
Cho KHT, Davidson JO, Dean JM, et al (2020) Cooling and immunomodulation for treating hypoxic-ischemic brain injury. Pediatrics International 62:770–778. https://doi.org/10.1111/ped.14215
Davidson JO, Wassink G, van den Heuij LG, et al (2015) Therapeutic Hypothermia for Neonatal Hypoxic–Ischemic Encephalopathy – Where to from Here? Frontiers in Neurology 6:. https://doi.org/10.3389/fneur.2015.00198
Davies A, Wassink G, Bennet L, et al (2019) Can we further optimize therapeutic hypothermia for hypoxic-ischemic encephalopathy? Neural Regeneration Research 14:1678–1683. https://doi.org/10.4103/1673-5374.257512
de Fraga LS, Tassinari ID, Jantsch J, et al (2021) “A picture is worth a thousand words”: the use of microscopy for imaging neuroinflammation. Clinical & Experimental Immunology 00:1–21. https://doi.org/10.1111/cei.13669
Dhikav V, Anand K (2012) Hippocampus in health and disease: An overview. Annals of Indian Academy of Neurology 15:239. https://doi.org/10.4103/0972-2327.104323
Escartin C, Galea E, Lakatos A, et al (2021) Reactive astrocyte nomenclature, definitions, and future directions. Nature Neuroscience 24:312–325. https://doi.org/10.1038/s41593-020-00783-4
Fabres RB, da Rosa LA, de Souza SK, et al (2018) Effects of progesterone on the neonatal brain following hypoxia-ischemia. Metabolic Brain Disease 1–9. https://doi.org/10.1007/s11011-018-0193-7
Fabres RB, Montes NL, Camboim Y de M, et al (2020) Long-Lasting Actions of Progesterone Protect the Neonatal Brain Following Hypoxia-Ischemia. Cellular and Molecular Neurobiology. https://doi.org/10.1007/s10571-020-00827-0
Fang AY, Gonzalez FF, Sheldon RA, Ferriero DM (2013) Effects of combination therapy using hypothermia and erythropoietin in a rat model of neonatal hypoxia-ischemia. Pediatric Research 73:12–17. https://doi.org/10.1038/pr.2012.138
Gunn AJ, Gunn TR, De Haan HH, et al (1997) Dramatic neuronal rescue with prolonged selective head cooling after ischemia in fetal lambs. Journal of Clinical Investigation 99:248–256. https://doi.org/10.1172/JCI119153
Haim L Ben, Carrillo-de Sauvage MA, Ceyzériat K, Escartin C (2015) Elusive roles for reactive astrocytes in neurodegenerative diseases. Frontiers in Cellular Neuroscience 9:1–27. https://doi.org/10.3389/fncel.2015.00278
Hermans RHM, Hunter DE, Mcgivern RF, et al (1992) Behavioral sequelae in young rats of acute intermittent antenatal hypoxia. Neurotoxicology and Teratology 14:119–129. https://doi.org/10.1016/0892-0362(92)90060-N
Heyser CJ (2003) Assessment of Developmental Milestones in Rodents. Current Protocols in Neuroscience 25:1–15. https://doi.org/10.1002/0471142301.ns0818s25
Jatana M, Singh I, Singh AK, Jenkins D (2006) Combination of systemic hypothermia and N-acetylcysteine attenuates hypoxic-ischemic brain injury in neonatal rats. Pediatric Research 59:684–689. https://doi.org/10.1203/01.pdr.0000215045.91122.44
Joly LM, Mucignat V, Mariani J, et al (2004) Caspase Inhibition after Neonatal Ischemia in the Rat Brain. Journal of Cerebral Blood Flow and Metabolism 24:124–131. https://doi.org/10.1097/01.WCB.0000100061.36077.5F
Lehmann J, Pryce CR, Bettschen D, Feldon J (1999) The Maternal Separation Paradigm and Adult Emotionality and Cognition in Male and Female Wistar Rats. Pharmacology Biochemistry and Behavior 64:705–715. https://doi.org/10.1016/S0091-3057(99)00150-1
Li J, Feng L, Xing Y, et al (2014) Radioprotective and antioxidant effect of resveratrol in hippocampus by activating Sirt1. International Journal of Molecular Sciences 15:5928–5939. https://doi.org/10.3390/ijms15045928
Liddelow SA, Guttenplan KA, Clarke LE, et al (2017) Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541:481–487. https://doi.org/10.1038/nature21029
Lubics A, Reglodi D, Tamás A, et al (2005) Neurological reflexes and early motor behavior in rats subjected to neonatal hypoxic-ischemic injury. Behavioural Brain Research 157:157–165. https://doi.org/10.1016/j.bbr.2004.06.019
Mari C, Odorcyk FK, Sanches EF, et al (2019) International Journal of Developmental Neuroscience Arundic acid administration protects astrocytes , recovers histological damage and memory de fi cits induced by neonatal hypoxia ischemia in rats. International Journal of Developmental Neuroscience 76:41–51. https://doi.org/10.1016/j.ijdevneu.2019.06.003
Mestriner RG, Pagnussat AS, Boisserand LSB, et al (2011) Skilled reaching training promotes astroglial changes and facilitated sensorimotor recovery after collagenase-induced intracerebral hemorrhage. Experimental Neurology 227:53–61. https://doi.org/10.1016/j.expneurol.2010.09.009
Nelson KB, Lynch JK (2004) Stroke in newborn infants. Lancet Neurology 3:150–158. https://doi.org/10.1016/S1474-4422(04)00679-9
Netto CA, Sanches E, Odorcyk FK, et al (2017) Sex-dependent consequences of neonatal brain hypoxia-ischemia in the rat. Journal of Neuroscience Research 95:409–421
Nie X, Lowe DW, Rollins LG, et al (2016) Sex-specific effects of N-acetylcysteine in neonatal rats treated with hypothermia after severe hypoxia-ischemia. Neuroscience Research 108:24–33. https://doi.org/10.1016/j.neures.2016.01.008
Owoeye O, Malomo AO (2015) Launaea taraxacifolia leaf extract protected against gamma radiation – induced haematological , behavioural and histological alterations in the hippocampus and cerebellum of rats. archives Basic & Appied Medicine 3:21–28
Park WS, Sung SI, Ahn SY, et al (2015) Hypothermia augments neuroprotective activity of mesenchymal stem cells for neonatal hypoxic-ischemic encephalopathy. PLoS ONE 10:1–13. https://doi.org/10.1371/journal.pone.0120893
Paxinos G, Watson C (2007) The Rat Brain in Stereotaxic Coordinates, Elsevier S. SanDiego, CA.
Reddy SD, Wu X, Kuruba R, et al (2020) Magnetic resonance imaging analysis of long‐term neuropathology after exposure to the nerve agent soman: correlation with histopathology and neurological dysfunction. Annals of the New York Academy of Sciences 1–20. https://doi.org/10.1111/nyas.14431
Reinboth BS, Köster C, Abberger H, et al (2016) Endogenous hypothermic response to hypoxia reduces brain injury: Implications for modeling hypoxic-ischemic encephalopathy and therapeutic hypothermia in neonatal mice. Experimental Neurology 283:264–275. https://doi.org/10.1016/j.expneurol.2016.06.024
Sabir H, Osredkar D, Maes E, et al (2016) Xenon combined with therapeutic hypothermia is not neuroprotective after severe hypoxia-ischemia in neonatal rats. PLoS ONE 11:1–10. https://doi.org/10.1371/journal.pone.0156759
Sabir H, Scull-Brown E, Liu X, Thoresen M (2012) Immediate hypothermia is not neuroprotective after severe hypoxia-ischemia and is deleterious when delayed by 12 hours in neonatal rats. Stroke 43:3364–3370. https://doi.org/10.1161/STROKEAHA.112.674481
Sabir H, Walløe L, Dingley J, et al (2014) Combined treatment of Xenon and hypothermia in newborn rats - Additive or synergistic effect? PLoS ONE 9:1–7. https://doi.org/10.1371/journal.pone.0109845
Saegusa Y, Fujimoto H, Woo GH, et al (2012) Transient aberration of neuronal development in the hippocampal dentate gyrus after developmental exposure to brominated flame retardants in rats. Archives of Toxicology 86:1431–1442. https://doi.org/10.1007/s00204-012-0824-4
Saegusa Y, Woo GH, Fujimoto H, et al (2010) Sustained production of Reelin-expressing interneurons in the hippocampal dentate hilus after developmental exposure to anti-thyroid agents in rats. Reproductive Toxicology 29:407–414. https://doi.org/10.1016/j.reprotox.2010.03.006
Sanches EF, Arteni NS, Nicola F, et al (2013) Early hypoxia-ischemia causes hemisphere and sex-dependent cognitive impairment and histological damage. Neuroscience. https://doi.org/10.1016/j.neuroscience.2013.01.066
Schneider T, Przewłocki R (2005) Behavioral alterations in rats prenatally to valproic acid: Animal model of autism. Neuropsychopharmacology 30:80–89. https://doi.org/10.1038/sj.npp.1300518
Shankaran S, Laptook AR, Ehrenkranz RA, et al (2005) Whole-Body Hypothermia for Neonates with Hypoxic–Ischemic Encephalopathy. New England Journal of Medicine 353:1574–1584. https://doi.org/10.1056/NEJMcps050929
Sheikhbahaei S, Morris B, Collina J, et al (2018) Morphometric analysis of astrocytes in brainstem respiratory regions. J Comp Neurol 526:2032–2047. https://doi.org/10.1002/cne.24472
Sizonenko S V., Camm EJ, Dayer A, Kiss JZ (2008) Glial responses to neonatal hypoxic-ischemic injury in the rat cerebral cortex. International Journal of Developmental Neuroscience 26:37–45. https://doi.org/10.1016/j.ijdevneu.2007.08.014
Smith A, Garbus H, Rosenkrantz T, Fitch R (2015) Sex Differences in Behavioral Outcomes Following Temperature Modulation During Induced Neonatal Hypoxic Ischemic Injury in Rats. Brain Sciences 5:220–240. https://doi.org/10.3390/brainsci5020220
Smith AL, Rosenkrantz TS, Fitch RH (2016) Effects of sex and mild intrainsult hypothermia on neuropathology and neural reorganization following neonatal hypoxic ischemic brain injury in rats. Neural Plasticity 2016:. https://doi.org/10.1155/2016/2585230
Sofroniew M V (2015) Astrogliosis. Cold Spring Harbor Perspectives in Biology 7:a020420. https://doi.org/10.1101/cshperspect.a020420
Soltesz I, Losonczy A (2018) CA1 pyramidal cell diversity enabling parallel information processing in the hippocampus. Nature Neuroscience 21:484–493. https://doi.org/10.1038/s41593-018-0118-0
Sun H-S, Xu B, Chen W, et al (2015) Neuronal KATP channels mediate hypoxic preconditioning and reduce subsequent neonatal hypoxic–ischemic brain injury. Experimental Neurology 263:161–171. https://doi.org/10.1016/j.expneurol.2014.10.003
Tassinari IDÁ, Andrade MKG, da Rosa LA, et al (2020) Lactate Administration Reduces Brain Injury and Ameliorates Behavioral Outcomes Following Neonatal Hypoxia–Ischemia. Neuroscience. https://doi.org/10.1016/j.neuroscience.2020.09.006
Thoresen M, Bågenholm R, Løberg EM, et al (1996) Posthypoxic cooling of neonatal rats provides protection against brain injury. Archives of Disease in Childhood 74:0–5. https://doi.org/10.1136/fn.74.1.f3
Thoresen M, Hobbs CE, Wood T, et al (2009) Cooling combined with immediate or delayed xenon inhalation provides equivalent long-term neuroprotection after neonatal hypoxia-ischemia. Journal of Cerebral Blood Flow and Metabolism 29:707–714. https://doi.org/10.1038/jcbfm.2008.163
Thoresen M, Tooley J, Liu X, et al (2013) Time Is Brain: Starting Therapeutic Hypothermia within Three Hours after Birth Improves Motor Outcome in Asphyxiated Newborns. Neonatology 104:228–233. https://doi.org/10.1159/000353948
Wagner BP, Nedelcu J, Martin E (2002) Delayed postischemic hypothermia improves long-term behavioral outcome after cerebral hypoxia-ischemia in neonatal rats. Pediatric Research 51:354–360. https://doi.org/10.1203/00006450-200203000-00015
Wood T, Osredkar D, Puchades M, et al (2016) Treatment temperature and insult severity influence the neuroprotective effects of therapeutic hypothermia. Scientific Reports 6:23430. https://doi.org/10.1038/srep23430
Xiong M, Yang Y, Chen G, Zhou W (2009) Post-ischemic hypothermia for 24 h in P7 rats rescues hippocampal neuron : Association with decreased astrocyte activation and inflammatory cytokine expression. Brain research bulletin 79:351–357. https://doi.org/10.1016/j.brainresbull.2009.03.011
Yuan X, Ghosh N, McFadden B, et al (2015) Hypothermia modulates cytokine responses after neonatal rat hypoxic-ischemic injury and reduces brain damage. ASN Neuro 6:. https://doi.org/10.1177/1759091414558418

GraphicAbstract.tif

Download PDF

Reviews received at journal
09 Mar, 2022
Reviewers invited by journal
09 Mar, 2022
Editor assigned by journal
03 Mar, 2022
First submitted to journal
06 Feb, 2022

You are reading this latest preprint version

Therapeutic hypothermia for the treatment of neonatal hypoxia-ischemia: sex-dependent modulation of reactive astrogliosis

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Animals

2.2 Neonatal hypoxia-ischemia

2.3 Therapeutic Hypothermia

2.4 Body temperature and body weight

2.5 Histological analysis

2.6 Cell counting

2.7 Immunofluorescence and morphological analysis

2.8 Behavioural Assessment

2.9 Data analysis

3. RESULTS

3.1 Body weight

3.2 Brain injury volume

3.3 Pathology score

3.4 Percentage of degenerative cells

3.5 GFAP immunoreactivity and astrocyte morphology

3.6 Behavioral Assessment

3.7 Correlation Analysis

4. Discussion

5. Conclusion

Declarations

Funding:

Competing interests:

Data availability statement:

Ethics approval:

References

Supplementary Files

Status:

Version 1