Therapeutic hypothermia has emerged as the first treatment to provide neuroprotection in neonatal HIE in multiple randomized controlled trials (3–5). A variety of mechanisms have been implicated in the effects of hypothermia, including cell death inhibition, cerebral metabolic rate reduction, and diminished release of excitotoxins and reactive oxygen species (6). As reviewed in a recent meta-analysis, hypothermia was shown to significantly reduce mortality and neurodevelopmental disability at 18 mo in human neonates cooled to 33–35 °C for 72 h within the first 6 h of life (5). The rationale for human trials emerged from animal studies of perinatal asphyxia, notably in lamb and piglet models, in which early initiation of hypothermia improved histological and functional outcomes (7,8). Brief hypothermia provided partial neuroprotection, whereas prolonged hypothermia (for 24–72 h) improved long-term behavioral performance (9).

Although hypothermia has been adopted as standard of care in many clinical units for infants with HIE (10), it does not provide complete neuroprotection, prompting urgent investigation into additional agents that may further improve outcomes. One such agent is erythropoietin (Epo), which has recently been shown to preserve brain structure and function in several studies of rodent neonatal stroke (11–13) and some studies of rodent HI (14–16). Although Epo was originally identified for its role in erythropoiesis, it has since been shown to have a wide range of functions that may account for its neuroprotective effects, including cell death inhibition, immune response modulation, initiation of angiogenesis, and promotion of neurogenesis, thereby enhancing repair (17). In human neonates with HIE, large-scale trials have yet to be performed, but some early clinical studies have demonstrated the safety and benefit of Epo treatment at 18 mo (18,19).

Although both hypothermia and Epo show great potential as treatments for neonatal HIE, there is much controversy in the literature regarding optimization of these treatments in the neonatal rodent HI model. For hypothermia, a wide variety of target temperatures, ranging from 22 to 35 °C, and durations, from 3 to 72 h, have been evaluated with mixed results (20–22). Similarly, optimal dosing and timing of Epo treatment in the neonatal rodent HI model remains under investigation, with most studies showing maximum benefit with doses between 1,000 and 5,000 U/kg administered in a multidose regimen between three and five times (15,16,23). In addition, sex-specific differences in treatment response to both hypothermia and Epo have been observed (24,25). Using a rodent model of neonatal HI, we evaluated the in vivo effects of whole-body cooling to 32 °C and multidose Epo administered at 1,000 U/kg and assessed whether combination therapy with both agents would improve functional and structural outcomes beyond either treatment alone.

Epo has previously been shown to improve histology and function after neonatal rodent stroke when using a similar treatment protocol (13). However, this regimen was not beneficial in this study’s model of neonatal HI and may have used a suboptimal dosage or number of doses. The literature regarding the usage of Epo in neonatal rodent HI is quite heterogeneous; different timing and dosage of Epo have had variable results. For example, a single dose of Epo at 1,000 U/kg was shown to improve short- and long-term brain injury and behavioral outcomes (14). Other studies reported benefits of Epo when administered in multiple doses of 500–1,000 U/kg, with no further benefit observed at 2,000 U/kg (16). Another study comparing high-dose Epo protocols reported benefit with doses ranging from 5,000 to 30,000 U/kg, with most benefit arising from multiple doses of 5,000 U/kg (23). Of note, some studies demonstrate improvement in sensorimotor outcomes without significantly improving histopathological outcomes. For example, a recent study showed that delayed and repeated dosing of 1,000 U/kg improved sensorimotor performance and enhanced oligodendrogliosis, but was ineffective at decreasing infarct volume (26). Similarly, three daily doses of 2,500 U/kg improved functional outcomes into adulthood without improving histopathology (27). Recently, enhanced benefit was noted with a nano-derivative of erythropoietin in a neonatal rat stroke model, which may facilitate blood–brain barrier crossing (28). Indeed, the optimal regimen of treatment with Epo in this model remains to be elucidated, as the ideal dosage, number of doses, and timing of administration are still not clear.

In addition, no observable benefit from hypothermia was noted in this study. Of note, although rats at age P7–P10 are often used to model the term newborn brain, P7 may actually represent a more immature (late-preterm) brain, and P10 may more accurately represent the full-term brain (29). In fact, earlier studies have shown equivocal benefit with target temperatures ranging from 31 to 34 °C for a total of 3 h (8), with only Thoresen et al. (30) showing improvement in histopathological outcomes when cooling to 32 °C rectal temperature for 3 h. Bona et al. (24) reported that cooling for 6 h following HI resulted in improved brain injury scores and sensorimotor outcomes in female animals. More recent studies have focused on longer duration of hypothermia, with mixed results. Hypothermia for 10 h to 32 °C and 26 °C did not improve striatal damage (22), whereas others demonstrated short-term histological improvements with the same duration of hypothermia to 30 °C (31). Even longer duration of hypothermia has been evaluated, with histological and functional improvement following 24–48 h of hypothermia to 30 °C (20) except in severely injured animals (32). Although 72 h of cooling to 33 °C has become standard of care for human neonates with suspected HIE, the optimal protocol for hypothermia in the neonatal rat model of HI has, like Epo dosing, yet to be completely identified. It is possible that more benefit can be gained from deeper or more prolonged hypothermia.

Other methodological issues have been raised regarding the administration of hypothermia in rodents. Most studies induce hypothermia via temperature-controlled ambient air temperatures or by titration of ambient air temperature to reach a target rectal temperature. In our model, hypothermia was administered with air cylinders partially submerged in a water bath adjusted to 32 °C, whereas normothermic animals were kept in air cylinders submerged in a 37 °C water bath. Mean cranial skin temperature of hypothermic animals was ~31 °C, whereas that of normothermic animals kept was 34 °C, raising the issue of whether normothermic animals were actually kept in mildly hypothermic conditions. A preferable strategy may have been titration of water bath temperature to maintain ambient air temperature within cylinders.

An additional concern regarding hypothermic temperature regulation involves the stress response to cooling. In this experiment, we found a significant difference in blood glucose following temperature treatment not noted in previous studies (24), raising the issue of potential compensatory responses to cooling that might interfere with neuroprotection, such as agitation, tachycardia, and shivering. Many studies demonstrated hypothermic neuroprotective effects in humans and piglets administered cooling under intensive-care settings, including anesthesia. A notable study in piglets demonstrated that lack of anesthesia during cooling resulted in higher stress levels and abolished the neuroprotective benefit of hypothermia (33). Indeed, there may even be detrimental effects from unanesthetized hypothermia that we were unable to detect in this study. In addition, the use of morphine during hypothermia in humans has been shown to be neuroprotective synergistically (34). In light of these studies, potential improvements to the current experimental protocol may include sedative and analgesic administration.

In contrast with some previous studies, no correlation between histological injury and sensorimotor performance was observed (11,13). Treated animals did not differ from untreated animals or sham-treated animals in forepaw preference in the cylinder-rearing test, making interpretation of treatments difficult. It is unclear why there was a lack of correlation between histology and function, although the heterogeneity of injury patterns seen here in HI animals, with mild and severely injured subjects mixed with relatively few moderately injured animals, may cloud the results. Although such a wide range of injury has been historically reported with this model (35), the high incidence of severe damage may represent the inaccuracy of this rodent model in determining short-term sensorimotor outcomes. There is probably a threshold for damage beyond which no treatment is beneficial (27,32), as well as a target group with moderate damage that will likely benefit the most from treatment. The effects of therapy may be more pronounced if animals with moderate injury can be identified and selected for treatment, as performed in other studies using magnetic resonance imaging (20). In addition, it is also possible that the cylinder-rearing test was insufficiently sensitive to detect differences between groups, or that all HI-injured animals regardless of injury severity were able to functionally compensate in this assay. Other tests of sensorimotor and cognitive function, and testing at later time points, may better demonstrate differences between treated groups. Finally, population-level differences in paw preference may also have obscured results, because sham-treated animals in this study appeared to have a right-paw preference that has not been previously reported in other studies.

Analysis of sex-based differences in outcome was performed in response to many studies in the literature that report sex-specific neuroprotective effects for a number of therapies, including hypothermia (24) and Epo (25,36). In this study, we found improvement in histopathological outcomes after the treatment with Epo that was specific to male animals. Brain development is known to be dimorphic and influenced by circulating hormones and differential gene expression even in the absence of hormonal influences (37), and this likely underlies sex differences in brain injury and response to therapies, although the specifics of these effects remain largely unknown (38). Neither sex-specific effects on sensorimotor outcomes in the different experimental groups after multifactorial analysis nor sex-specific differences on histopathological outcome in animals that received hypothermia alone or combination therapy were observed.