The only current treatment for neonatal hypoxia-ischemia (HI) is therapeutic hypothermia (TH), which still shows some limitations. Specific effects of TH in the several processes involved in brain injury progression remain unclear. In this study, the effects of TH treatment on developmental parameters, behavioral outcomes, and peripheral leukocytes were evaluated in neonatal male and female rats. In P7, animals were submitted to right common carotid artery occlusion followed by hypoxia (8% oxygen). TH was performed by reducing the animal scalp temperature to 32°C for 5 h. Behavioral parameters and developmental landmarks were evaluated. Animals were euthanized at P9 or P21, and cerebral hemispheres, spleen, and thymus were weighed. White blood cells (WBCs) were counted in blood smears. There was a reduction in the weight of the brain hemisphere ipsilateral to the carotid occlusion in HI and TH groups, as well as a reduction in body weight gain and a delay in the opening of the ipsilateral eye. Latency in negative geotaxis was increased by HI at P12. TH did not prevent brain weight loss, developmental impairments, or WBC number changes but prevented negative geotaxis impairment and spleen weight reduction. These data reinforce that a better understanding of the events that occur after HI and TH in both males and females is necessary and would allow the development of more adequate and sex-specific therapeutic approaches.

Hypoxia-ischemia (HI) is a major cause of neonatal death and neuropsychological deficiencies, such as cognitive deficit, visual and hearing impairments, epilepsy, and cerebral palsy [1, 2]. Therapeutic hypothermia (TH), the only treatment currently available for newborns after HI, consists in lowering the newborn’s body temperature to 33.5°C, decreasing the metabolic rate and consequently the brain injury [3]. However, some limitations in the use of TH have been reported: TH has limited efficacy in patients with severe brain injury [4, 5], is not recommended for preterm babies [6], and must be initiated within 6 h after the hypoxic-ischemic event [4]. The existence of such limitations demands more studies on the effects of TH for the treatment of neonatal HI.

Brain injury is the main consequence of HI, varying among different brain areas and according to sex [7]. Preferential apoptosis pathways differ between males and females [8, 9] and males tend to show poorer overall conditions after HI [7, 10]. Different levels of neuroprotection provided by TH are also observed when comparing sexes [11‒13] and females may benefit more from the effects of TH treatment in an overall perspective [14, 15]. Neuroinflammation is one of the processes that participates in the formation of brain injury. The contribution of peripheral immunity in these processes is well established in models of stroke in adult experimental animals [16, 17]. Although some studies show that this can also play an important role in neonatal HI [18], it has been less studied in the immature immune system of neonates. Invasion of peripheral immune cells into the site of brain lesion could contribute to the onset and extension of neuroinflammation [19].

Besides brain injury, impairments in developmental parameters and behavioral outcomes show peripheral impacts of HI [20, 21], which also show sexually dimorphic responses. Males display mainly motor deficits, whereas females show mainly memory-related deficits [22]. However, most behavioral tests used in neonatal rodents, such as righting reflex and negative geotaxis, aim to assess the motor and sensory capability of the animal [23]. The olfactory discrimination test used here can be an alternative of behavioral assessment of cognitive function in neonatal rats [24‒26].

The aim of this study was to evaluate the impact of TH treatment on neurodevelopmental and peripheral immunity parameters in neonatal rats submitted to HI and determine whether the effects of TH are sexdependent. We hypothesized that the TH treatment reduces brain damage, and developmental and behavioral impairments, as well as reduces the activation of the peripheral immune system in neonatal rats submitted to HI.

Animals

A total of 168 Wistar rats (84 male and 84 female) at the 7th postnatal day (P7) were used in the present study. Pups were obtained from the Animal Breeding Center (CREAL, Centro de Reprodução e Experimentação de Animais de Laboratório) of the Federal University of Rio Grande do Sul (UFRGS) and kept with their dams in the Animal Facility (UEA, Unidade de Experimentação Animal) of the Hospital de Clínicas de Porto Alegre (HCPA), the University Hospital of UFRGS. Dams received food and water ad libitumand were maintained under a 12/12-h light/dark cycle at a temperature around 22°C. The study was approved by the Institutional Animal Care and Use Committee of HCPA (#2019-0420) and by the Research Committee of the Institute of Basic Health Sciences of UFRGS (#38998).

Experimental Design

Pups were randomly assigned to each of the following experimental groups: SHAM, HI, and TH. Animals from TH and HI groups were submitted to HI (described below) and underwent 5-h hypothermia (TH group; scalp temperature around 32°C) or normothermia (HI group; scalp temperature around 37°C). SHAM animals underwent fictitious surgery and were kept in normoxia and normothermia. The experimental design is detailed in Figure 1a.

Fig. 1.

Timeline of the experimental design (a) and scalp temperature measured every 15 min with an infrared thermometer in males (b) and females (c) during 5 h of TH or normothermia. Results are presented as mean ± SEM (n = 25–27). NG, negative geotaxis; OD, olfactory discrimination.

Fig. 1.

Timeline of the experimental design (a) and scalp temperature measured every 15 min with an infrared thermometer in males (b) and females (c) during 5 h of TH or normothermia. Results are presented as mean ± SEM (n = 25–27). NG, negative geotaxis; OD, olfactory discrimination.

Close modal

HI Procedure and Hypothermia Protocol

HI procedure was conducted according to the model of Rice-Vannucci [27] and our previous studies [28‒32]. Seven-day-old rats were anesthetized with isoflurane (5% for induction and 3% for maintenance), and a small longitudinal incision was performed around 2 mm to the right of the trachea followed by divulsion of the muscles to reach the right common carotid artery. Next, the right common carotid artery was isolated and permanently occluded with a surgical silk thread. After surgery, pups returned to their dams for 75 min for recovery and breastfeeding. Then, 4–6 animals were placed inside a chamber and exposed to a hypoxic atmosphere (8% oxygen) for 75 min at 33°C. To maintain the adequate body temperature of the pups (37°C), the chamber was kept within a neonatal incubator (Fanem C186TS). Animals from the SHAM group were submitted to fictitious surgery (without carotid occlusion) and kept in normoxia. After hypoxia, the pups returned to their dams for 90 min and then were submitted to hypothermia into a neonatal incubator (Fanem C186TS) for 5 h, maintaining the scalp temperature around 32°C (Fig. 1b and c) [5, 31]. Scalp temperature was measured every 15 min using an infrared thermometer (TCI1000 Incoterm) to avoid contact with the animals [32]. Animals from SHAM and HI groups were kept in another incubator (Fanem C186TS) for 5 h maintaining their scalp temperature at 37°C (normothermia). After 5 h of hypothermia, animals from the TH group were re-warmed on a heating table for no less than 20 min until scalp temperature reached 37°C and then returned to their dams.

Brain Weight

Brains were harvested at P9 and P21. Both brain hemispheres (right, ipsilateral to carotid occlusion and left, contralateral) were separated and weighed apart. The weight ratio of ipsilateral to contralateral hemispheres was calculated as ipsilateral weight/contralateral weight × 100. This value was used as an estimation of the percentage of tissue loss in the cerebral hemisphere ipsilateral to carotid occlusion [33, 34].

Developmental Milestones

At P7 and once every 2 days after P8, animals were weighed with a precision balance (Marte AS5500c). Animals euthanized at P9 or that died before reaching P21 were excluded from body weight analysis. During weighing (every 2 days), it was observed if the opening of the eyes (complete separation of the eyelids) had occurred, and the first observation was registered for each eye separately [35].

Behavioral Assessment

Animals were tested at four different ages (behavioral tests are described below). All behavioral tests were performed at P8, P10, P12, and P14 and conducted in the afternoon. The animals’ performances were recorded and analyzed later.

Negative Geotaxis

Animals were placed head down on the surface of a 35° inclined plane, and the latency to perform a 180° turn was recorded (maximum of 60 s). Each animal was tested once [23].

Olfactory Discrimination Test

In an acrylic box (40 × 20 × 20 cm), soiled bedding from the home cage was placed on one side of the box, whereas on the opposite side, clean bedding was placed on the box surface. Beddings were spread in a rectangular area of about 12 cm in length from the lateral wall to the center of the box and 20 cm in length along the two extremities of the box. Each animal was placed in the center of the cage, facing the front wall, and the latency to reach one of the beddings with the four paws was measured (maximum of 120 s) [36]. Each pup was tested twice (positions of the home and clean beddings were switched for each test, i.e., positions relative to the left or right side of the animals). The box was cleaned with 70% ethanol before each test.

Lymphoid Organs Weight

At P9 or P21, the spleen and thymus were collected and immediately weighed. The percentage of the organ weight relative to the body weight was calculated by the formula: organ weight/body weight × 100.

White Blood Cell Counting

The trunk blood was collected after euthanasia in tubes containing EDTA. Blood smears were prepared, fixated in methanol 100% for 30 s [37], and stained using hematological stain (Panótico Rápido, Laborclin). For each animal, 2 blood smears were prepared. The slides were used for total white blood cell (WBC) counting, using a ×20 objective, in 10 defined fields in the tail region of the blood smear [38].

Statistical Analysis

For an overall analysis of body weight, negative geotaxis, and olfactory discrimination test, curves were constructed for each animal, from which the respective areas under the curve (AUCs) were calculated in order to verify the existence of differences between groups over the experimental period. Two-way ANOVA followed by Sidak was used to evaluate negative geotaxis, spleen and thymus weight, with group and sex as factors. Kruskal-Wallis test followed by Dunn was used to analyze brain and body weights, the olfactory discrimination test, and WBC counting. Mann-Whitney U test was used for the analysis of the day of eye opening (comparison between right and left sides). All statistical analyses were performed using the SPSS PASWStat v.18 and Prism® GraphPad 8.0.2 software. Data were presented as mean ± standard error of the mean or median, interquartile ranges (25th and 75th percentile), and minimum and maximum values. Values of p < 0.05 were considered significant.

Mortality Rate

From 168 animals utilized in this study, 4 died during surgery. From the 117 allocated in the HI or TH groups, 5 animals (3 from the HI and 2 from the TH group) died in the days following HI exposure, resulting in a mortality rate of 4.27%.

Brain Weight

Representative images of the harvested brains are shown in Figure 2a. For both sexes, the presence of cerebral edema was seen in the right hemisphere (ipsilateral to carotid occlusion) in the HI and TH groups in P9; the edema was absent in P21, but a striking tissue loss could be observed.

Fig. 2.

Representative images from harvested brains (a) and weight ratio of right to left (ipsilateral and contralateral to carotid occlusion, respectively) brain hemispheres at P9 (b) and P21 (c). Body weight for males (d) and females (e) along with the respective area under the curve (AUC) (f). Body weight curves are presented as mean ± SEM; AUC and brain weight ratios are represented as median and interquartile range (25th and 75th percentiles) as boxes and minimum and maximum values as whiskers. *p < 0.05 versus SHAM, Kruskal-Wallis followed by Dunn (P9: n = 11–12; P21: n = 14–16). L, left; R, right; Ro, rostral; Cd, caudal. Scale bar: 0.5 cm.

Fig. 2.

Representative images from harvested brains (a) and weight ratio of right to left (ipsilateral and contralateral to carotid occlusion, respectively) brain hemispheres at P9 (b) and P21 (c). Body weight for males (d) and females (e) along with the respective area under the curve (AUC) (f). Body weight curves are presented as mean ± SEM; AUC and brain weight ratios are represented as median and interquartile range (25th and 75th percentiles) as boxes and minimum and maximum values as whiskers. *p < 0.05 versus SHAM, Kruskal-Wallis followed by Dunn (P9: n = 11–12; P21: n = 14–16). L, left; R, right; Ro, rostral; Cd, caudal. Scale bar: 0.5 cm.

Close modal

In P9, HI and TH showed a reduced brain weight ratio in comparison to SHAM in males and only relative to TH animals in females (H(5) = 25.49; p < 0.001) (Fig. 2b). In P21, animals from the HI and TH groups presented a reduced brain weight ratio when compared to SHAM animals, regardless of sex (H(5) = 56.44; p < 0.001) (Fig. 2c). This indicates that there was a brain weight loss in the hemisphere ipsilateral to the carotid occlusion in the animals from the HI and TH groups. Also, this weight loss was more pronounced at P21 (approximately 40%) than at P9 (approximately 15%).

Developmental Milestones

As expected, animals from all experimental groups gained body weight over time (Fig. 2d, males; Fig. 2e, females). However, there was a significant effect of the factor “group” (F(2,84) = 42.88; p < 0.001) as a smaller AUC was seen in animals from HI and TH groups when compared to SHAM (Sidak, p < 0.001) (Fig. 2f).

There was a significant delay in the day of the right eye (ipsilateral to carotid occlusion) opening when compared to the left eye (contralateral to carotid occlusion) of the same animal in the HI group (Table 1) (males: U = 43.50; p < 0.001; females: U = 29.50; p < 0.001), as well as in the TH group (males: U = 25.50; p < 0.001; females: U = 24.00; p < 0.001) for both sexes. When compared to SHAM animals, it was observed that pups from HI and TH groups showed a delay in right eye opening in females and only in the TH group in males (H(5) = 35.74; p < 0.001). The day of the left eye opening showed no difference among experimental groups (H(5) = 6.02; p = 0.304). We have also noted that, even when eye opening occurred, some animals of the HI and TH groups maintained the right eye only partially open or had a white spot in the center of the right eye, probably indicating an eye injury not evaluated here.

Table 1.

Day of eye opening of the right and left eyes in male and female animals

Experimental groupMalesFemales
rightleftrightleft
SHAM 14.0 (14.0–14.0) 14.0 (14.0–14.0) 14.0 (14.0–14.0) 14.0 (14.0–14.0) 
HI 16.0 (14.0–18.0)a 14.0 (14.0–14.0) 18.0 (14.5–21.0)*, a 14.0 (14.0–14.0) 
TH 16.0 (16.0–20.0)*, a 14.0 (14.0–14.0) 20.0 (16.0–21.0)*, a 14.0 (14.0–14.0) 
Experimental groupMalesFemales
rightleftrightleft
SHAM 14.0 (14.0–14.0) 14.0 (14.0–14.0) 14.0 (14.0–14.0) 14.0 (14.0–14.0) 
HI 16.0 (14.0–18.0)a 14.0 (14.0–14.0) 18.0 (14.5–21.0)*, a 14.0 (14.0–14.0) 
TH 16.0 (16.0–20.0)*, a 14.0 (14.0–14.0) 20.0 (16.0–21.0)*, a 14.0 (14.0–14.0) 

Values are presented as median with interquartile range (25th and 75th percentiles).

*Differences related to the same eye of SHAM animals (p < 0.05 in Kruskal-Wallis, followed by Dunn).

aDifferences related to the left eye of the same group (p < 0.05 in Mann-Whitney U test) (n = 14–16).

Negative Geotaxis

The latency of the animals in the negative geotaxis test for each age evaluated is shown in Figure 3a (males) and b (females). In P8, P10, and P14, no significant differences among the experimental groups were verified. In P12, however, there was an effect of the factor “group” (F(2,84) = 3.67; p = 0.030) as the HI group showed a higher latency to complete the test as compared to the SHAM group (Sidak, p = 0.028). No significant difference was observed between the TH and SHAM groups (Sidak, p = 0.199). The overall performance of male and female animals in the test was assessed by the evaluation of the AUC (Fig. 3c), but no significant differences between the experimental groups were verified.

Fig. 3.

Latency to perform neurological reflexes tests and respective area under the curve (AUC) in negative geotaxis (a–c) and latency to climb with the four paws on the home bedding in the olfactory discrimination test, when the home bedding was on the right side (d, e) or the left side (g, h) of the animal, with the respective areas under the curve (AUCs) (f, i). All behavioral tests were performed at P8, P10, P12, and P14 in male (a, d, g) and female (b, e, h) animals. Results are presented as mean ± SEM, except for f and i which are represented as median and interquartile range (25th and 75th percentiles) as boxes and minimum and maximum values as whiskers. *p < 0.05 versus SHAM, two-way ANOVA followed by Sidak (n = 12–18).

Fig. 3.

Latency to perform neurological reflexes tests and respective area under the curve (AUC) in negative geotaxis (a–c) and latency to climb with the four paws on the home bedding in the olfactory discrimination test, when the home bedding was on the right side (d, e) or the left side (g, h) of the animal, with the respective areas under the curve (AUCs) (f, i). All behavioral tests were performed at P8, P10, P12, and P14 in male (a, d, g) and female (b, e, h) animals. Results are presented as mean ± SEM, except for f and i which are represented as median and interquartile range (25th and 75th percentiles) as boxes and minimum and maximum values as whiskers. *p < 0.05 versus SHAM, two-way ANOVA followed by Sidak (n = 12–18).

Close modal

Olfactory Discrimination Test

The results of the olfactory discrimination test are shown in Fig. 3d–i. No significant differences between the experimental groups were observed in any of the ages or in the AUC of both home bedding positioning trials.

White Blood Cell Count

For the WBC counting at P9 (Fig. 4a), no differences were observed in males, but HI and TH females had a reduction in WBC count in comparison to SHAM females (H(5) = 26.79; p < 0.001). Also, HI males had a greater WBC count in comparison to HI and TH females. At P21 (Fig. 4b), HI and TH males had a reduction in WBC count when compared to SHAM males (H(5) = 49.79; p < 0.001). No differences were observed between female groups, but HI females had a higher WBC count in comparison to HI males.

Fig. 4.

White blood cell (WBC) counting from blood smears in male and female animals at P9 (a) and P21 (b). Spleen weight (c, d) and thymus weight (e, f) relative to body weight in male and female pups at P9 (c, e) and P21 (d, f). WBC counting results are presented as median and interquartile range (25th and 75th percentiles) as boxes and minimum and maximum values as whiskers; spleen and thymus weight results are presented as mean ± SEM; *p < 0.05 versus SHAM, two-way ANOVA followed by Sidak or Kruskal-Wallis followed by Dunn (WBC, P9: n = 240; P21: n = 120–140; spleen and thymus weight, P9: n = 11-12; P21: n = 14–16).

Fig. 4.

White blood cell (WBC) counting from blood smears in male and female animals at P9 (a) and P21 (b). Spleen weight (c, d) and thymus weight (e, f) relative to body weight in male and female pups at P9 (c, e) and P21 (d, f). WBC counting results are presented as median and interquartile range (25th and 75th percentiles) as boxes and minimum and maximum values as whiskers; spleen and thymus weight results are presented as mean ± SEM; *p < 0.05 versus SHAM, two-way ANOVA followed by Sidak or Kruskal-Wallis followed by Dunn (WBC, P9: n = 240; P21: n = 120–140; spleen and thymus weight, P9: n = 11-12; P21: n = 14–16).

Close modal

Weight of Lymphoid Organs

There was no significant difference in the weight of the spleen in P9 (Fig. 4c). At P21, there was an effect of the factor “group” (F(2,84) = 4.44; p = 0.015) as the HI group showed a reduction in spleen weight compared to the SHAM group (Sidak, p = 0.016), but the TH group did not (Sidak, p = 0.093) (Fig. 4d). The weight of the thymus in P9 also showed an effect of the factor “group” (F(2,63) = 3.74; p = 0.029) as it was reduced in the TH groups in comparison with the SHAM groups (Sidak, p = 0.025) (Fig. 4e), whereas in P21 no effects were observed (Fig. 4f).

Several studies show the beneficial effects of TH, the standard treatment for HI insults in human newborns since 2010 [3]. Here, we also showed the advantageous effects of TH, preventing behavioral impairments and spleen weight reduction after HI. However, we also found that the hypothermic treatment was limited in preventing brain weight loss, developmental impairments, and WBC count changes. It is noteworthy that this is one of the few studies that evaluated the behavior of the animals for several days following HI.

Our results showed a consistent unilateral brain injury caused by HI, which was more pronounced at P21. It is well known that brain injury increases during the days following HI, mainly due to the activation of pro-inflammatory pathways [39]. In P9, a greater edema formation was observed soon after HI, while in P21, there was a notable tissue loss but absence of edema. Indeed, previous studies showed that edema is present mainly in the earlier stages of HI [40‒42].

The reduction in the brain weight observed here in the HI and TH groups is thought to be related to tissue loss after HI, as previously observed [33, 34, 43]. However, TH was not able to revert this decrease in brain weight caused by HI. Some studies have shown that TH is most effective when the injury is moderate and less effective in reducing brain damage in cases of severe injury [4, 5]. Furthermore, TH efficacy remains unclear regarding mild injury [44]. Here, brain weight loss was approximately 15% in P9 and 40% in P21, which could be equivalent to the percentage of tissue loss observed histologically in cases of mild brain injuries [4, 5].

Besides producing a brain lesion, HI also negatively affected the development of the pups, as previously described by others: animals exposed to HI show a reduction in the body weight gain [12, 21, 28, 29, 45] and a delay in the opening of the eye ipsilateral to carotid occlusion [46, 47]. Interestingly, these developmental impairments were not ameliorated by TH treatment. Matsuda et al. [48] observed that hypothermia per se (in control animals), when applied to 30-h-old rats (preterm model), had negative effects on developmental parameters, causing a delay in the neurodevelopment. This interesting finding may help to explain the reason why TH was not effective in preventing some of the negative effects of HI seen here. In the present study, however, we used animals at the age of P7. The P7 rat model of HI used here has been considered equivalent to a late preterm human newborn [49]. The clinical use of TH is currently contraindicated for preterm human neonates due to possible negative developmental effects [50]. In animal models, negative effects of hypothermia were already observed in pups at P6 or earlier ages [51], but positive effects on white matter injury were also seen [52]. Conversely, after HI induction in rats at P10-12, age currently considered as a near-term model, TH shows positive results [49]. Similarly, both beneficial and negative effects of the treatment were also observed in the P7 rats [32, 53, 54]. It is known that both HI and TH effects show high variability at this age [13]. Taken together, these are possible explanations of the absence of beneficial effects of TH in reducing detrimental effects of HI, especially considering the brain weight loss, and add more evidence to the variability of the treatment, suggesting caution in interpreting the results. Moreover, it is worth mentioning that variations in the experimental protocols, such as cooling depth, duration, and delay to start hypothermia, affect differently neuroprotection and the evaluated outcomes (e.g., volume of brain injury vs. behavioral responses).

A study by Sabir et al. [4], for example, found that 10 h of hypothermia does not confer additional neuroprotection when compared to 5 h. However, the use of mild hypothermia even for shorter periods showed beneficial results if hypothermia is applied during hypoxia exposure (not after hypoxia) [11]. Here, we showed that 5 h of hypothermia can show beneficial or inefficient results depending on the outcome evaluated.

When animals were assessed in the negative geotaxis test, a greater latency in the HI group was found at the age of P12, showing a motor impairment in these animals, as seen by others [29, 45, 55‒57]. However, the other ages evaluated, and the AUC analyses were not affected by HI. As the brainstem, an important structure involved in this neurological reflex [58], receives its blood supply from vertebral arteries, it is supposed to be less affected by HI, which could prevent striking impairments in the negative geotaxis response. Studies that induce brain injury in rats younger than 7 days show that the injury can cause a delay in the appearance of neurological reflexes [21, 48]. Here, the exposure of the animals to HI at P7, a moment near reflex arousal, could have been less harmful to this behavioral response. Behavioral impairments are variable according to the brain injury severity, age of HI induction, and ages evaluated. Additional studies using different behavioral tests are still necessary to better understand the behavioral outcomes following HI and TH.

Unlike negative geotaxis, which involves motor and sensory components, the olfactory discrimination test also requires learning and decision-making, being one of the earlier cognitive tests that can be used in rat pups [24‒26]. This olfactory behavior has been evaluated only in a few studies, which observed that HI increased the latency to reach the home bedding [59, 60]. However, here, HI and TH did not affect this behavior. Moreover, to the best of our knowledge, this is the first study to use the olfactory discrimination test to assess the effects of TH following HI.

WBC counting, another parameter evaluated here, also showed a time- and sex-dependent response. In males, a reduction in the number of leukocytes in HI and TH groups was observed in P21, suggesting later immunosuppression caused by HI, which has been observed in human patients, leading to secondary infections [61], and has been observed in experimental animal models as well [62, 63]. Females showed an earlier decrease in WBC counting in HI and TH groups, but in P21, it returned to control levels. It seems that the changes in this parameter occur at different times according to sex.

Also, a reduction in spleen weight was observed at P21, leading us to hypothesize that the spleen is contracting and releasing cells to the circulation at this period [64]. Previous studies showed a reduction in spleen weight and number of immune cells, accompanied by an increase in the number of immune cells invading the brain [17, 65]. Thymus weight was reduced in TH animals at P9. Only one study in the literature showed thymus atrophy after occlusion of the medial cerebral artery [65] and another showed an increase in the apoptosis of thymic cells after stroke [66], both in adult rats. In neonates, thymus plays an important role in the maturation of T cells [67, 68]. T cells infiltrate the brain 3 days after HI and increase brain damage [69]. On the other hand, the anti-inflammatory actions of Treg cells can exert neuroprotective effects [69]. More studies on thymus and T cells roles after HI are necessary to understand inflammatory progression and also possible effects of TH treatment.

Some limitations in this study can be highlighted. We evaluated the brain injury by weighing the brain, but this analysis is less precise, especially in the presence of an edema. This could be masking beneficial effects of TH on brain injury. Also, the variability of outcomes after HI and TH treatment [13] is a factor that demands caution in the interpretation of the results. Regarding the peripheral immune parameters, there is also a lack of evaluation of molecular mechanisms involved in inflammatory pathways.

In summary, our data indicate that TH, despite its several beneficial effects and efficacy in preventing neonatal deaths worldwide, should be used with caution during the perinatal period. The efficacy of TH in preventing brain injury and the developmental impairments showed limitations in the present study. However, TH was beneficial on behavioral parameters and prevented spleen weight reduction. It reinforces the need to better understand the events that occur after HI in both males and females, the mechanisms underlying TH effects, and the search for therapeutic strategies that are complementary to TH.

This study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of HCPA (approval number 2019-0420) and by the Research Committee of the Institute of Basic Health Sciences of UFRGS (approval number 38998). The study was conducted in accordance with the ARRIVE guidelines.

The authors have no conflict of interest to declare.

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundo de Incentivo à Pesquisa e Eventos from Hospital de Clínicas de Porto Alegre (FIPE/HCPA).

R.R. Nunes, I.D. Tassinari, and J. Zang designed the study, acquired, analyzed, and interpreted the data, and drafted the initial version of the manuscript. M.K.G. Andrade, A.C.M. Colucci, M.L.M. Hoff, and M.R. de Oliveira contributed to data collection and analysis. A.H. Paz and L.S. de Fraga conceptualized and designed the study and contributed to funding acquisition, project administration and supervision, data interpretation, and review of the manuscript. All the authors critically revised the manuscript and approved the submitted version.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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