Sex‐Dependent Long‐Term Neurobehavioral Recovery in Experimental Neonatal Hypoxic–Ischemic Encephalopathy

ABSTRACT

This study investigates the sex‐dependent effects of therapeutic hypothermia and a neuroprotectant (CHF6467) on long‐term neurobehavioral recovery in neonatal rats subjected to hypoxic–ischemic encephalopathy. Using the Rice–Vannucci model, rats were divided into five groups: sham lesion, hypoxic–ischemic insult, hypoxic–ischemic insult with CHF6467, hypoxic–ischemic insult with hypothermia, and hypoxic–ischemic insult with CHF6467 combined with hypothermia. Behavioral tests at postnatal day 30 (P30) and 60 (P60) revealed significant improvements in motor coordination and spatial memory in treated groups compared to the vehicle‐treated group, associated with reductions in infarct size at P60. Notably, female rats exhibited superior motor coordination recovery compared to males, despite similar neuroprotection levels. This suggests that motor coordination recovery is not solely dependent on the extent of neuronal damage. Conversely, spatial memory impairment was sex‐independent and closely associated with the degree of neuroprotection. These findings highlight the importance of considering sex differences in neuroprotective treatment efficacy and suggest potential variations in responses between males and females in clinical settings.

Therapeutic hypothermia and CHF6467 reduce brain injury and improve outcomes in rats after neonatal hypoxia‐ischemia. Long‐term motor recovery was superior in female animals and significantly inversely associated with brain lesion size.

1. Introduction

Therapeutic hypothermia (TH) is the standard of care for reducing morbidity and mortality in neonates with moderate‐to‐severe hypoxic–ischemic encephalopathy (HIE) [1]. A key question is whether there are sex‐dependent differences in response to TH. While recent large clinical studies have reported greater susceptibility of males to hypoxia–ischemia, no significant sex differences were observed in the subgroup of infants treated with TH [2]. This may be explained by the broad mechanism of action of TH, which targets multiple cell death pathways and provides similar neuroprotection in both male and female subjects [3]. An important unresolved question is whether the response to novel neuroprotective treatments in combination with hypothermia could reveal sex‐related differences. This aspect should be investigated using experimental animal models to inform future clinical research.

Indeed, sex may influence the efficacy of neuroprotective interventions, including TH, in rodent models of neonatal HIE, with overall evidence suggesting greater benefits in females than in males [4]. This variability partly arises from the commonly used Rice–Vannucci model, which involves unilateral carotid ligation and induced hypoxia at postnatal day 7 (P7), followed by either normothermia or hypothermia. The reasons for the apparent discrepancies in sex‐dependent effects of TH across animal species remain unclear. One possible explanation for the absence of notable sex differences in studies involving large animals is that research conducted on piglets and fetal sheep may have lacked sufficient statistical power to detect these differences.

To help bridge the gap between preclinical and clinical observations, we conducted a retrospective analysis of a relatively large histological and behavioral study on TH, a derivative of human nerve growth factor (CHF6467) [5], and their combination in rats subjected to the Rice–Vannucci model [6]. The study by Landucci et al. was originally designed to assess the histopathological and behavioral efficacy of CHF6467 in neonatal rats subjected to hypoxic–ischemic insult and was not specifically powered to evaluate sex differences in treatment response. Rats were randomly allocated to treatment groups, and all measurements were performed under blinded conditions. Behavioral testing and brain infarct analyses were conducted concurrently.

CHF6467, also referred to as “painless human NGF” (hNGFp), was developed following clinical observations of hereditary sensory autonomic neuropathy type V—a rare genetic disorder caused by a point mutation at position 100 of NGF (R100W). This mutation results in a normal phenotype with markedly reduced pain perception [5]. CHF6467 incorporates a similar mutation at position 100 (R100E) to reduce its algogenic activity, along with an additional mutation at position 61 (P61S) to allow analytical distinction from endogenous wild‐type NGF. Intranasal administration of CHF6467 in a mouse model of Alzheimer's disease was shown to differentially modulate microglia and astroglia by regulating tumor necrosis factor‐alpha expression and enhancing amyloid‐β clearance, thereby reducing amyloid plaque burden [7].

2. Methods

The methods of the study have been previously described in detail [6]. All surgical and experimental procedures were carried out in accordance with the Italian regulations for the care and use of laboratory animals (EU Directive 2010/63/EU) and were approved by appropriate institutional and state authorities of University of Florence (no. 22/2022‐PR). Briefly, pregnant Wistar rats were housed individually under a regular light/dark cycle. The day of delivery was designated as postnatal day 0 (P0). On P7, Wistar rat pups were anesthetized with isoflurane, and the left common carotid artery was permanently ligated via a midline neck incision. Following the procedure, pups were returned to their dams for 1 h to recover and nurse. Subsequently, they were placed in enclosed, ventilated Plexiglas chambers partially submerged in a water bath and exposed to a continuous flow of warmed, humidified gas (8% oxygen, 92% nitrogen) for 120 min. After hypoxia, pups were returned to their dams for 1 h, then placed in chambers maintained at either normothermic (37°C) or hypothermic (32°C) temperatures for 4 h. The animals were randomly assigned to five groups: 23 rats undergoing a sham lesion, 21 rats undergoing hypoxic–ischemic insult, 20 rats undergoing hypoxic–ischemic insult and receiving CHF6467, 19 rats undergoing hypoxic–ischemic insult and receiving hypothermia, and 20 rats undergoing hypoxic–ischemic insult and receiving CHF6467 combined with hypothermia. At P7, pups were administered CHF6467 (20 μg/kg) intranasally immediately after hypoxia and again at 24, 48, and 72 h post‐hypoxia. The pups were then maintained with their dams until weaning and subjected to behavioral tests at P30, equivalent to early adolescence, and at P60, equivalent to late human adolescence. Motor coordination was assessed using the Rotarod test (latency to fall), and spatial memory was evaluated using the Y‐maze test (percent alternations). After behavioral testing, the animals were sacrificed, and brain infarct sizes were estimated using toluidine blue staining. Brain infarct size, latency to fall, and percent alternations were analyzed using a two‐way analysis of variance (ANOVA), with treatment, gender, and their interaction as fixed factors, and rats as a random factor. Comparisons were performed relative to the vehicle‐treated lesioned group using the Holm‐Sidak procedure. Statistical associations between brain infarct size, latency to fall, and percent alternations were assessed using Pearson's product–moment correlation test. Statistical comparisons of the Pearson's correlation coefficients between brain infarct size, latency to fall, and percent alternations were performed using Fisher's r‐to‐z transformation test.

3. Results

At P30, hypoxic–ischemic rats remained on the Rotarod for a significantly shorter time compared to the sham‐lesioned group (−86%). This effect was significantly attenuated by treatment with hypothermia, CHF6467 alone, or their combination (Figure 1, left panel). In the Y‐maze test, the hypoxic–ischemic group showed a significantly reduced percent alternations compared to sham‐lesioned animals (−20%), and this reduction was completely reversed by hypothermia, CHF6467 alone, or their combination (Figure 1, right panel). No significant differences were observed between male and female animals in either latency to fall or percentage of alternations. These behavioral data obtained at P30 were generated during the study described by Landucci et al. [6] and have not been previously published. The animals were subsequently behaviorally tested at P60, and the outcome is reported by Landucci et al. [6].

FIGURE 1.

Behavioral performance of 103 rats on the Rotarod (left panel) and Y‐maze (right panel) measured at postnatal day 30 in sham‐lesioned animals (n = 23) and in hypoxic–ischemic animals treated at postnatal day 7 with vehicle (n = 21), hypothermia (n = 19), CHF6467 (n = 20), or hypothermia + CHF6467 (n = 20). Hypoxic–ischemic damage was induced at postnatal day 7. *p < 0.01 versus vehicle‐treated animals.

At P60, the mean (±SEM) infarct volumes were 103.7 ± 11.3, 39.3 ± 10.3, 44.8 ± 10.3, and 18.2 ± 7.5 mm³ for the vehicle, hypothermia, CHF6467, and hypothermia + CHF6467 groups, respectively. Both hypothermia and CHF6467, as well as their combination, significantly reduced brain infarct size compared to the vehicle‐treated group (p < 0.001). We did not detect a significant effect of gender or a gender‐by‐treatment interaction on brain infarct size. On the Rotarod, the mean latency to fall was 30.2 ± 5.0, 212.7 ± 27.8, 251.3 ± 24.6, and 279.5 ± 22.4 s for the vehicle, hypothermia, CHF6467, and hypothermia + CHF6467 groups, respectively. We found significant effects of treatment (p < 0.001), gender (p < 0.001), and treatment‐by‐gender interaction (p = 0.007) on latency to fall. In the vehicle group, male (n = 27) and female (n = 53) animals did not differ in Rotarod performance. However, female rats treated with hypothermia (p < 0.001), CHF6467 (p = 0.002), or hypothermia + CHF6467 (p = 0.011) had significantly longer latencies to fall compared to male rats. In the Y‐maze test, we observed a significant effect of treatment (p < 0.001) on percent alternations but no significant effect of gender (p = 0.091) or treatment‐by‐gender interaction (p = 0.436). We also found a significant inverse association between brain infarct volume and latency to fall (r = −0.522, p < 0.001) and percent alternations (r = −0.445, p < 0.001).

Interestingly, although the correlation between brain damage and spatial memory was similar in female and male animals (Figure 2 upper), the association between brain infarct size and motor coordination on the Rotarod was significant in female rats (r = −0.537, p < 0.001) but not in male rats (r = −0.330, p = 0.129; Figure 2 lower). However, the difference between the Pearson correlation coefficients for female and male animals was not statistically significant (p = 0.242).

FIGURE 2.

Scatter plot of brain infarct volume and percent alternation on the Y‐maze test in 53 female (upper left panel) and 27 male (upper right panel) rats measured at postnatal Day 60 treated with vehicle (n = 21), hypothermia (n = 19), CHF6467 (n = 20), and hypothermia + CHF6467 (n = 20) at postnatal day 7. Scatter plot of brain infarct volume and latency to fall on the Rotarod test in 53 female (lower left panel) and 27 male (lower right panel) rats measured at postnatal day 60 in 80 rats treated with vehicle (n = 21), hypothermia (n = 19), CHF6467 (n = 20), and hypothermia + CHF6467 (n = 20) at postnatal Day 7. Hypoxic–ischemic damage was induced at postnatal day 7. Pearson correlation coefficients and associated p values are shown, as well as linear regression lines and associated 95% confidence intervals.

4. Discussion

This retrospective analysis shows a significant effect of sex on long‐term recovery of motor coordination following hypothermia or a neuroprotectant and of their combination in rats exposed to hypoxic–ischemic brain injury at 7 days of age. Female animals performed, on average, approximately 70% better than male animals following hypothermia, intranasal CHF6467, or their combination, despite the extent of neuroprotection being nearly identical between the two genders. This indicates that, in hypoxic–ischemic rats, the recovery of motor coordination is not solely dependent on the extent of neuronal damage. In contrast, spatial memory impairment in hypoxic–ischemic rats appears to be sex‐independent and closely associated with the extent of neuroprotection. A study in neonatal rats subjected to hypoxic–ischemic brain damage at P10 and evaluated at P70 found that female animals exhibited a more pronounced serum anti‐inflammatory response (interleukin‐10) and enhanced neurogenesis (bromo‐2′‐deoxyuridine incorporation into neurons) compared to male rats [8].

There is a well‐recognized difference in the incidence and outcome of neonatal encephalopathy according to sex, with a pronounced male disadvantage. Neurodevelopmental differences manifest from early infancy, with females showing a lower incidence of developmental delay and learning difficulties compared to males. Male sex has also been consistently identified as a risk factor for cerebral palsy [9]. Several potential mechanisms may underlie these sex differences, including genetic, immunological, and hormonal factors. Animal models of neonatal HIE have demonstrated various sex‐related differences in behavioral performance, favoring female animals [9]. These models also show an upregulated immune response and increased microglial activation in males [9]. In contrast to the behavioral differences, postmortem neuropathology associated with neonatal hypoxia–ischemia appears comparable between sexes [10]. These findings suggest that the relative protection observed in females in certain behavioral domains may be due to sex‐specific plasticity or compensatory mechanisms, rather than a reduced burden of gross neuropathological damage.

The results of our study confirm and expand upon previous research indicating that TH leads to sex‐specific improvements in motor function in rat models of neonatal HIE [11]. It is unclear why female rats performed better than males after intranasal administration of CHF6467, despite similar levels of neuroprotection. However, it is recognized that inflammation—triggered by activation of both the central and peripheral immune systems—is a key pathogenic factor in neonatal brain injury [12]. Neuroinflammatory responses can be initiated within minutes of the insult and may persist for weeks or even months. Previous studies have shown that CHF6467 attenuates cytokine upregulation in brain tissue from neonatal rats subjected to hypoxic–ischemic injury [6]. CHF6467 may work by reducing neuroinflammation linked to hypoxic–ischemic brain injury, which could be especially beneficial in female animals, as their neuroinflammatory responses are generally milder and more responsive to treatment.

It is crucial to validate these findings in other experimental animal species to ensure that the observed relationship is not specific to this rodent model. A recent post hoc subgroup analysis of a study involving 101 infants with moderate or severe neonatal encephalopathy found no sex differences in the rate of death or moderate‐to‐severe disability at 18–22 months following whole‐body hypothermia [13]. However, long‐term studies conducted at 5–6 years of age may reveal sex‐dependent responses to TH on more detailed endpoints, such as neuromotor coordination, as suggested by our current rodent study.

Looking ahead, if the observed sex differences in motor coordination recovery from brain injury are confirmed in humans, additional treatments alongside hypothermia should account for potential variations in responses between males and females.

Author Contributions

Elisa Landucci: conceptualization, data curation, formal analysis, writing – original draft, writing – review and editing. Bruno P. Imbimbo: conceptualization, data curation, formal analysis, supervision, writing – original draft, writing – review and editing. Alessia Melani: resources. Domenico E. Pellegrini‐Giampietro: supervision, writing – review and editing. Fabrizio Facchinetti: conceptualization, supervision, writing – original draft, writing – review and editing.

Conflicts of Interest

Bruno P. Imbimbo and Fabrizio Facchinetti are employees of Chiesi Farmaceutici S.p.A. They do not hold any stocks or equity shares in Chiesi Farmaceutici S.p.A. Elisa Landucci, Alessia Melani, and Domenico E. Pellegrini‐Giampietro declare no conflicts of interest.

Landucci E., Imbimbo B. P., Melani A., Pellegrini‐Giampietro D. E., and Facchinetti F., “Sex‐Dependent Long‐Term Neurobehavioral Recovery in Experimental Neonatal Hypoxic–Ischemic Encephalopathy,” Pharmacology Research & Perspectives 13, no. 5 (2025): e70183, 10.1002/prp2.70183.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.