Cerebral palsy (CP) is the most common cause of physical disability for children worldwide. Many infants and toddlers are not diagnosed with CP until they fail to achieve obvious motor milestones. Currently, there are no effective pharmacologic interventions available for infants and toddlers to substantially improve their trajectory of neurodevelopment. Because children with CP from preterm birth also exhibit a sustained immune system hyper-reactivity, we hypothesized that neuro-immunomodulation with a regimen of repurposed endogenous neurorestorative medications, erythropoietin (EPO) and melatonin (MLT), could improve this trajectory. Thus, we administered EPO + MLT to rats with CP during human infant-toddler equivalency to determine whether we could influence gait patterns in mature animals. After a prenatal injury on embryonic day 18 (E18) that mimics chorioamnionitis at ∼25 weeks human gestation, rat pups were born and raised with their dam. Beginning on postnatal day 15 (P15), equivalent to human infant ∼1 year, rats were randomized to receive either a regimen of EPO + MLT or vehicle (sterile saline) through P20. Gait was assessed in young adult rats at P30 using computerized digital gait analyses including videography on a treadmill. Results indicate that gait metrics of young adult rats treated with an infantile cocktail of EPO + MLT were restored compared to vehicle-treated rats (p < 0.05) and similar to sham controls. These results provide reassuring evidence that pharmacological interventions may be beneficial to infants and toddlers who are diagnosed with CP well after the traditional neonatal window of intervention.

Cerebral palsy (CP) remains the most common cause of physical disability in children worldwide and is often associated with other disorders of sensation, cognition, communication, perception, and behavior [1, 2]. An estimated 50 million people live with CP [3]. While neonatal interventions are emerging for newborns at high risk of CP [4], less than half of children with CP have the diagnosis entertained as neonates. In addition, many infants and children often lack access to early diagnosis and resource-conscious interventions. Most babies at risk for CP in high-income countries do not receive their initial care at advanced neonatal centers that offer clinical trials for early diagnosis and treatment of central nervous system (CNS) injury. Importantly, the problem is worse in low- and middle-income countries, where children with CP have less access to both diagnosis and interventions, despite high rates of children with more severe motor impairment [5]. For example, CP contributes 5.7% of disability-adjusted life-years from neurological disorders in India [6]. Children with CP also suffer medical co-morbidities that increase utilization of health care resources [7]. Children and young adults with more severe CP are also likely to experience more pain, fatigue, and mental health issues [8], emphasizing the cascading effect of CP and its co-morbidities on lifelong function and independence. To this end, there is a pressing need for safe pharmacological interventions for infants and toddlers who miss the neonatal window for interventions. Indeed, the lack of proven, effective interventions to restore motor deficits after CP is diagnosed in young children that often drives families of children with few options to seek unproven, costly, and potentially dangerous treatments [9]. Moreover, recognition of the need for safe, effective, resource-conscious pharmacological interventions for infants at high risk of CP aligns with the growing international trend toward earlier diagnosis and intervention for CP using physical, occupational, speech, and cognitive therapeutic programs for infants and children [10‒16].

Spontaneous very preterm birth, often associated with chorioamnionitis, is a leading cause of CP [17, 18]. To test potential therapeutic strategies in a clinically relevant model of CP, we developed a rat model of chorioamnionitis, a major contributor to CNS injury and CP from preterm birth [19, 20]. Adult rats exposed to chorioamnionitis exhibit a spastic gait, as well as poor motor inhibition, social interaction, and cognition [20]. These rats also show sustained impairment of systemic immune regulation [21‒24], similar to children born preterm with CP [25]. Neonatal cocktail therapy with endogenous neurorestorative hormones erythropoietin (EPO) and melatonin (MLT) restored CNS development and function in this model of preterm brain injury, including gait, inhibition, social interaction, and executive function [20]. Notably, neither EPO nor MLT alone as monotherapy was able to restore functional deficits [20]. Subsequently, other groups replicated these results using neonatal cocktail therapy in a model of term hypoxic-ischemic encephalopathy (HIE) [26]. Both EPO and MLT possess established records of safety as monotherapy in clinical trials in neonates [27‒34]. EPO and MLT are known to be essential for neurodevelopment [35‒37]. Both are also known to optimize the CNS microenvironment by reducing inflammation, endoplasmic reticulum stress and mitochondrial injury, excessive protease activation, and reactive oxygen species [36, 38‒42]. EPO has been under investigation as a neonatal neurorestorative agent for decades [39, 43‒45]. While MLT is most well known for its receptor-mediated effects on circadian rhythm [46], the MLT effects in this cocktail are likely receptor independent [47‒49]. Notably, the exact mechanisms of the synergistic interaction of EPO plus MLT in the injured developing brain remain under active investigation. Given that EPO and MLT are developmentally regulated, it is not known if delayed infantile treatment would be effective for the neurorestoration of gait.

Due to the urgent need for pharmacological therapy for infants who are diagnosed with CP after the neonatal window of intervention, we tested here whether a cocktail therapy with EPO + MLT begun in infancy at postnatal day 15 (P15) could show efficacy. We hypothesized that infantile treatment using EPO + MLT could restore deficits in gait and posture in young adults using a rat model of CP secondary to chorioamnionitis.

Chorioamnionitis Model of CP

With approval from the Johns Hopkins Institutional Animal Care and Use Committee, a laparotomy was performed on pregnant Sprague-Dawley dams on embryonic day 18 (E18) under isoflurane anesthesia, per our prior work [19, 50]. Uterine arteries were transiently occluded for 60 min, followed by intra-amniotic injection of 4 μg of lipopolysaccharide (LPS, 0111:B4; Sigma). Sham controls received a laparotomy without transient uterine artery occlusion and LPS injection. All dams experienced an equivalent duration of anesthesia and laparotomy and perioperative medications. Pups were born at term and matured with their dams until weaning at P21.

Infantile Cocktail Therapy with EPO and MLT

On P1, pups of both sexes were coded to mask the injury and treatment group from observers. On P15, pups with prenatal exposure to chorioamnionitis were allocated randomly to either a regimen of infantile EPO + MLT or vehicle (equivalent volume of sterile saline) administered via intraperitoneal (i.p.) injection once daily from P15 through P20. The EPO + MLT regimen was based on a clinically relevant dosing strategy similar to prior studies [20]. Treated rats received EPO (2,000 U/kg/dose i.p. on P15, P17, and P19) and MLT (20/mg/kg/dose i.p. on P15–P20). Prior work has shown that this dosing regimen is well tolerated [20].

Computerized Digital Gait Analyses

On P30, multiple gait metrics were acquired and assessed with computerized digital gait analyses (Digigait, MouseSpecifics) by masked observers, per our prior publications [20, 41, 42]. Specifically, a transparent treadmill was set at 30 cm/s, and footprints digitally encoded. For each rat, 3 s of their typical running pattern was analyzed by the software. Rats who failed to run were retested (3 times total); 2 rats were excluded for failure to run. Because chorioamnionitis induces a global CNS injury, data from right and left hind paws were averaged for each rat. In sum, data from 45 shams (20 F, 25 M), 20 vehicle-treated rats with preterm brain injury (9 F, 11 M), and 13 EPO + MLT-treated rats with preterm brain injury (7 F, 6 M) were analyzed.

Statistical Analyses

Each variable represents the mean hind paw metric ± standard error of the mean. The same statistical methods were used to analyze data from male and female rats independently and male and female combined. Data were tested for normality with the Shapiro-Wilk test using Prism 9.3.0 (Graphpad). Group differences were compared with Kruskal-Wallis with Dunn’s post hoc correction (KW) for nonparametric data, and two-way ANOVA with Tukey’s post hoc correction (ANOVA) for parametric data, with p < 0.05 considered significant.

The spastic gait of CP is characterized by a shorter stride length, greater stride length variation, and higher step frequency in both ambulatory children with CP and rodent models [51]. Male and female rats within each group (sham, vehicle-treated preterm, and EPO + MLT-treated preterm) did not differ and were thus analyzed together. Vehicle-treated young adult rats with CNS injury from the preterm insult (n = 22) had a shorter stride length compared to rats treated with infantile EPO + MLT (n = 13, 2-way ANOVA, F(2, 77) = 4.71 p = 0.02), or sham controls (n = 45, p = 0.03, Fig. 1a). Vehicle-treated rats with CNS injury also exhibited more stride length variability than EPO + MLT-treated injured rats (KW statistic = 10.93, p = 0.009, Fig. 1b). Similarly, the vehicle-treated injured rats had a higher coefficient of variation for stride length than EPO + MLT-treated rats (KW statistic = 12.73, p = 0.005) or shams (p = 0.007, Fig. 1c). Vehicle-treated rats also had greater step frequency than injured rats treated with the infantile EPO + MLT regimen (KW statistic = 7.34, p = 0.042, Fig. 1d). Consistent with the shorter stride length and increased step frequency, the duration of the swing phase of gait was also significantly shorter in the vehicle-treated rats with brain injury compared to the EPO + MLT-treated rats (ANOVA, F(2, 76) = 6.25, p = 0.0457), or sham controls (p = 0.003, Fig. 1e). The infantile EPO + MLT regimen normalizes multiple gait parameters related to stride length in young adult rats with preterm brain injury, indicative of a durable effective therapy into young adulthood.

Fig. 1.

Gait of adult rats with preterm brain injury is reminiscent of a spastic gait in individuals with spastic CP. a Stride length is reduced in vehicle-treated rats with preterm brain injury compared to sham controls and EPO + MLT-treated rats with preterm brain injury. b Similarly, vehicle-treated rats with preterm brain injury exhibit more stride length variation than littermates treated with EPO + MLT. c Likewise, the coefficient of variation of stride length in vehicle-treated rats with preterm brain injury is more than in shams or EPO + MLT-treated rats with preterm brain injury. d Step frequency is higher in vehicle-treated rats compared to EPO + MLT-treated rats with preterm brain injury. e The swing phase of gait is shorter in vehicle-treated rats with preterm brain injury compared to shams and EPO + MLT-treated rats with preterm brain injury (*p< 0.05, **p< 0.01).

Fig. 1.

Gait of adult rats with preterm brain injury is reminiscent of a spastic gait in individuals with spastic CP. a Stride length is reduced in vehicle-treated rats with preterm brain injury compared to sham controls and EPO + MLT-treated rats with preterm brain injury. b Similarly, vehicle-treated rats with preterm brain injury exhibit more stride length variation than littermates treated with EPO + MLT. c Likewise, the coefficient of variation of stride length in vehicle-treated rats with preterm brain injury is more than in shams or EPO + MLT-treated rats with preterm brain injury. d Step frequency is higher in vehicle-treated rats compared to EPO + MLT-treated rats with preterm brain injury. e The swing phase of gait is shorter in vehicle-treated rats with preterm brain injury compared to shams and EPO + MLT-treated rats with preterm brain injury (*p< 0.05, **p< 0.01).

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In addition to a shorter stride length and more steps, the spastic gait of CP is also characterized by “toe-walking” with less “heel-to-toe” contact area and force on impact [51]. In this study, adult rats with preterm brain injury treated with vehicle showed reduced paw area at maximal contact compared to injured rats treated with the infantile EPO + MLT regimen (KW statistic = 10.66, p = 0.008, Fig. 2a). Similarly, the vehicle-treated rats with preterm brain injury also applied less pressure than rats treated with infantile EPO + MLT (KW statistic = 6.89, p = 0.029, Fig. 2b). These results show the infantile treatment with EPO + MLT also normalizes paw contact and thus improves gait efficiency in young adult rats with brain injury.

Fig. 2.

Rats with preterm brain injury exhibit toe-walking which is restored by infantile cocktail treatment with EPO + MLT. a Rats with preterm brain injury treated with vehicle contact showed significantly less paw area than EPO + MLT-treated littermates. b Similarly, vehicle-treated rats with preterm brain injury exert less pressure at maximal contact than EPO + MLT-treated rats (*p< 0.05).

Fig. 2.

Rats with preterm brain injury exhibit toe-walking which is restored by infantile cocktail treatment with EPO + MLT. a Rats with preterm brain injury treated with vehicle contact showed significantly less paw area than EPO + MLT-treated littermates. b Similarly, vehicle-treated rats with preterm brain injury exert less pressure at maximal contact than EPO + MLT-treated rats (*p< 0.05).

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Children with spastic CP also often exhibit ataxia and variable foot placement that impacts gait [51]. Vehicle-treated rats with preterm brain injury demonstrated a more variable paw angle than EPO + MLT-treated rats with brain injury (KW statistic = 8.49, p = 0.012, Fig. 3a). Injured rats treated with vehicle also showed marked ataxia compared to EPO + MLT-treated rats with preterm brain injury (KW statistic = 11.77, p = 0.008) or sham controls (p = 0.01, Fig. 3b). Together, these results show that a regimen of EPO + MLT administered to infantile-age rats can provide durable restoration of ataxia and hind paw alignment.

Fig. 3.

The unsteady, more variable gait pattern observed in adult rats with preterm brain injury treated with vehicle is restored with infantile EPO + MLT treatment. a Vehicle-treated rats with preterm brain injury show more deviations in paw angle than EPO + MLT-treated rats with preterm brain injury. b Likewise, vehicle-treated rats with preterm brain injury exhibit markedly more ataxia than EPO + MLT-treated littermates (*p< 0.05).

Fig. 3.

The unsteady, more variable gait pattern observed in adult rats with preterm brain injury treated with vehicle is restored with infantile EPO + MLT treatment. a Vehicle-treated rats with preterm brain injury show more deviations in paw angle than EPO + MLT-treated rats with preterm brain injury. b Likewise, vehicle-treated rats with preterm brain injury exhibit markedly more ataxia than EPO + MLT-treated littermates (*p< 0.05).

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To determine if both sexes responded to the infantile treatment with EPO + MLT, gait metrics were analyzed separately for males and females as a preliminary assessment. Similar to prior studies of preterm brain injury in Sprague-Dawley rats [39, 52], in this study male rats exhibited more severe deficits in gait metrics than females (Table 1). Overall, the study was not sufficiently powered with EPO + MLT-treated rats to fully assess the impact of sex on every functional outcome; however, a similar pattern in amelioration of gait metrics is apparent (Table 1). For example, vehicle-treated male rats exposed to prenatal chorioamnionitis (n = 12) exhibited more ataxia compared to EPO + MLT-treated male rats (n = 6, KW statistic = 8.61, p = 0.045) and sham male controls (n = 25, p = 0.028, Fig. 4a). By contrast, while female vehicle-treated rats with preterm brain injury (n = 10) showed a similar pattern of ataxia compared to female shams (n = 20), and female EPO + MLT-treated rats with preterm brain injury (n = 7), the trend was not significant (Fig. 4b). Overall, the severity of gait deficits from preterm brain injury was worse in males; however, the pattern of restoration of deficits by infantile EPO + MLT regimen was similar in young adult rats of both sexes (Table 1).

Table 1.

Gait metrics in males and females

 Gait metrics in males and females
 Gait metrics in males and females
Fig. 4.

Gait deficits from preterm brain injury are more pronounced in male rats and are normalized with infantile EPO + MLT treatment. a Male vehicle-treated rats with preterm brain injury show more ataxia than male shams or male EPO + MLT-treated rats with preterm brain injury (*p< 0.05). b Female vehicle-treated rats with preterm brain injury demonstrate a similar pattern, but the trend is not statistically significant.

Fig. 4.

Gait deficits from preterm brain injury are more pronounced in male rats and are normalized with infantile EPO + MLT treatment. a Male vehicle-treated rats with preterm brain injury show more ataxia than male shams or male EPO + MLT-treated rats with preterm brain injury (*p< 0.05). b Female vehicle-treated rats with preterm brain injury demonstrate a similar pattern, but the trend is not statistically significant.

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Many children with CP are not diagnosed until infancy when complex motor skills begin to emerge and well after the newborn period when current pharmacotherapies are undergoing testing in clinical trials. A huge unmet need exists for effective pharmacologic treatments for young children with CP to augment current interventions with physical and occupational therapy, botulinum toxin injections, and surgery [53]. Detailed gait evaluation using digital analyses provides a unique opportunity to rigorously test potential interventions. Gait deficits in preclinical models of CP were recently reviewed by da Conceicao Pereira and colleagues [54]. Their review suggests that preclinical models that combine components of hypoxia-ischemic and bacterial inflammation from LPS, such as in the current study, replicate the sustained gait deficits observed in many individuals with CP [54]. Interventions currently in clinical practice for newborns at risk of CP appear less effective in the setting of chronic inflammation, including prenatal magnesium for very preterm newborns [55], and hypothermia for term infants with HIE [56]. Thus, the inclusion of systemic inflammation is crucial to CNS injury models of CP. Prenatal chorioamnionitis on E18 induces durable alterations in gait and posture, with a shorter stride, more frequent steps, and more variability in juvenile, young adult, and adult animals [19, 20], reminiscent of children with a spastic gait in CP [51].

Few preclinical studies of infantile pharmacological interventions for neurorestoration in CP have been reported. Altamentova and colleagues [57] demonstrated that methylprednisolone administered on P21, equivalent to a 2-year-old toddler and 14 days after hypoxia-ischemia on P7, provided durable improvement in both histology and gait through young adulthood (P56). While methylprednisolone reduces neuroinflammation, broad immunosuppression during development may have secondary unintended consequences [58‒60], such as limiting the neurorestorative potential of the endogenous stem cell response that may be essential for replenishing lost neural cell progenitors [61]. Neuro-immunomodulation with endogenous neurorestorative hormones, such as EPO and MLT, may mitigate against unintended effects.

EPO and its mimetics have been tested in multiple clinical trials in the neonatal period to mitigate perinatal brain injury for preterm [27, 28, 34] and term infants [4]. While EPO as monotherapy restores gait [20, 39, 42, 62] and spatial memory [52] after preterm CNS injury in preclinical models, neither EPO alone nor MLT alone as monotherapy in a neonatal regimen improved motor inhibition nor cognitive flexibility in adult animals [20]. Notably, only a cocktail of neonatal EPO + MLT after preterm CNS injury improved executive function in adult animals [20], suggesting that the two endogenous hormones work in concert to restore cognition. Executive function and cognition are essential for optimizing rehabilitation strategies and later independence in children with CP as executive function is necessary for adherence to and consistent use of rehabilitative strategies. Executive functions are also critically important for independence in daily living.

Both EPO and MLT have well-established safety profiles when used as monotherapy in neonates [4, 27, 28, 34] and infants and children for different indications other than CP [63, 64]. While the safety profiles found in prior clinical experience offer reassurance, pharmacological interventions should only be tested within the framework of well-designed clinical trials, which can be challenging given the typical mixed etiologies encountered in children with CP. In addition, the variable etiology, clinical phenotype, and utilization of early interventions often compounds the complexity of clinical trial design for children with CP.

The cellular and molecular mechanisms of restoration of gait for CP using EPO + MLT regimens are currently under active investigation. Children who were born very preterm and have CP exhibit a persistent hyper-reactive immune system profile during childhood compared to children who were born very preterm but do not have CP [25]. Lin and colleagues’ [25] pivotal study demonstrating persistent alterations in immune function has subsequently been replicated in different populations with neurodevelopmental disorders, suggesting a strong correlation exists between immune system activation and healthy development and maturation of the CNS. For example, children with CP from neonatal HIE exhibit altered T-cell frequencies that persist through school-age [65], and children with CP have altered cytokine responses [66, 67]. Chronic systemic inflammation may disrupt endogenous neurorestorative processes in the CNS [68]. Importantly, the preclinical model of CP from preterm brain injury used in this study, in addition to exhibiting gait and cognitive deficits of children with CP from preterm birth [19, 20], demonstrates sustained peripheral immune hyper-reactivity [21‒24], reminiscent of the altered immune activation found in the clinical studies [25]. Recently, there has been greater recognition of the interplay between myeloid peripheral blood mononuclear cells (PBMCs, T and B lymphocytes, and monocytes) in meninges and CNS health [69‒71]. Human PBMCs express EPO receptors and are modulated by EPO [72]. Similarly, MLT exerts beneficial effects on T cells and immunomodulation in children with trisomy 21 [73, 74]. Thus, because both EPO and MLT are endogenous pleiotropic regulators of both the developing immune and neural systems, they are promising candidates for safe neuro-immunomodulation in young children with CP.

As monotherapies, EPO and MLT each promote healthy neural cell development and maintenance, as reviewed in the Introduction. As a cocktail, EPO and MLT may additively and/or synergistically drive pathways to augment sustained neuro-immunomodulation and promote microenvironmental homeostasis. Notably, levels of the nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase sirtuin 1 (silent mating-type information regulation 2 homolog 1 [SIR], SIRT1) are elevated by both EPO [75, 76] and MLT [77‒79] (Fig. 5). SIRT1 regulates metabolism and immune cell health via antioxidative, anti-inflammatory, and antiapoptotic effects [80, 81], and SIRT1 levels are reduced in PBMCs of preterm infants exposed to hyperoxia [82]. Additionally, SIRT1 shifts the hypoxia-inducible factor (HIF) switch from HIF-1α toward HIF-2α. During acute hypoxic challenges, the functions of HIF-1α and HIF-2α overlap [83‒85]. By contrast, in the setting of chronic inflammation, such as has been reported in children with CP [25, 65‒67], the functions of HIF-1α and HIF-2α diverge significantly. Specifically, during chronic inflammation HIF-1α drives toxic, fibrotic, pro-inflammatory responses while HIF-2α provides trophic, anti-inflammatory support [84, 85]. Notably, HIF-2α is also critical for immune cell health [86‒89]. Moreover, HIF-2α is the transcription factor that increases astrocytic production of EPO in the CNS [90] to sustain neuronal and oligodendrocyte health [35, 40]. Importantly, EPO requires adequate SIRT1 levels to exert its protective effects [76, 91], including neurorestorative effects following CNS injury [75]. Thus, EPO and MLT may act in concert to prevent SIRT1 deficiency and allow EPO to support optimal recovery in the injured developing brain. Healthy oligodendroglial lineage development and myelination are necessary for motor learning and myelin plasticity [92, 93]. In addition to the direct impact on neural cells, treatment with EPO + MLT may also influence immune cell health. More detailed studies of the proposed synergistic molecular mechanisms of EPO + MLT are underway and beyond the scope of this work. The initial functional improvement observed with infantile EPO + MLT treatment shown in this study supports this further investigation.

Fig. 5.

Potential mechanism for synergistic efficacy between EPO and MLT during extended infantile treatment for gait deficits. By increasing SIRT1, EPO and MLT drive the HIF switch toward HIF-2α and suppress HIF-1α. HIF-2α exerts multiple trophic effects including suppressing systemic hepcidin and ROS and inflammation in immune cells. In the developing CNS, HIF-2α also promotes astrocytic production of EPO, potentiating the impact of exogenous EPO for neural cell health, including neurons, microglia, and oligodendrocytes. ROS, reactive oxygen species.

Fig. 5.

Potential mechanism for synergistic efficacy between EPO and MLT during extended infantile treatment for gait deficits. By increasing SIRT1, EPO and MLT drive the HIF switch toward HIF-2α and suppress HIF-1α. HIF-2α exerts multiple trophic effects including suppressing systemic hepcidin and ROS and inflammation in immune cells. In the developing CNS, HIF-2α also promotes astrocytic production of EPO, potentiating the impact of exogenous EPO for neural cell health, including neurons, microglia, and oligodendrocytes. ROS, reactive oxygen species.

Close modal

This study has limitations. First, the study is not sufficiently powered to adequately distinguish differences in potential benefit due to sex. Assessment of the effect of sex in larger cohorts of male and female rats will be the subject of future studies on putative sexual dimorphism in therapeutic response. Second, the current study has not addressed the potential cognitive benefits of EPO + MLT treatment during infancy; these studies are underway. Third, we have not yet detailed the neuro-immunomodulation impact on immune cells after this regimen of infantile EPO + MLT treatment. Given that the neural immune system is altered through adulthood in this model of preterm brain injury [24], we will be pursing this line of investigation. In addition to investigating the mechanisms of neuro-immunomodulation, we will correlate outcomes with advanced imaging and fluid biomarkers to guide clinical trial design. The goal of this initial study was to test whether delayed modulation of the immune and neural systems until during infancy could sufficiently alter the trajectory of neurodevelopment after prenatal injury and restore gait deficits in adult animals. Now that we have demonstrated initial functional benefit from an infantile EPO + MLT regimen, we will pursue additional studies. Notably, the developmental equivalences stated herein are based on overall neural development, specifically oligodendrocyte maturation concomitant with cortical layer maturation, immune system development, and integrity of central excitatory and inhibitory circuitry. This is important as this study was done in rats and we acknowledge important species differences between rats and humans with respect to weight bearing, locomotion, and the developmental trajectory of motor and spinal circuit maturation.

In conclusion, in this study we demonstrated using multiple gait metrics in a preclinical model of CP that the window for efficacy of neuroreparative pharmacological regimens to restore adult motor function extends well into infancy. These studies greatly extend the potential opportunity to treat a much larger proportion of infants and toddlers diagnosed with CP, as well as potentially those identified as at high risk for CP. These results support more investigation in preparation for potential clinical trials to optimize early brain development using widely available repurposed medications with well-established safety profiles. Importantly, such pharmacologic strategies could be integrated with early PT/OT/cognitive therapy interventions to maximize developmental outcomes. Finally, here we tested infantile treatment in a model of CP from preterm brain injury secondary to chorioamnionitis. Given the extensive overlap in mechanisms of impaired neurodevelopment from related neurodevelopmental insults and syndromes, potential pharmacologic interventions during infancy could have widespread impact on children’s health.

This study protocol was reviewed and approved by Johns Hopkins Institutional Animal Care and Use Committee, approval numbers RA19M401 (Shenandoah Robinson) and RA21M364 (Lauren Jantzie).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The authors are grateful for the generous funding provided by the Cerebral Palsy Alliance Research Foundation (CPARF) to Lauren Jantzie, Frances Northington, and Shenandoah Robinson and the National Institutes of Health (NIH) R01HL139492 to Lauren Jantzie.

Conceptualization and design: Shenandoah Robinson and Lauren Jantzie; methodology: Shenandoah Robinson and Lauren Jantzie; investigation: Sankar Muthukumar, Yuma Kitase, Vikram Vasan, Mohammed Fouda, Sarah Hamimi, Christopher Burkardt, Vera Joanna Burton, Gwendolyn Gerner, and Joseph Scafidi; formal analysis, Xiaobu Ye, Shenandoah Robinson, Frances Northington, Vera Joanna Burton, Gwendolyn Gerner, Joseph Scafidi, and Lauren Jantzie; writing – original draft preparation: Shenandoah Robinson and Lauren Jantzie; writing – review and editing: Lauren Jantzie, Sankar Muthukumar, Yuma Kitase, Vikram Vasan, Mohammed Fouda, Sarah Hamimi, Christopher Burkhardt, Vera Joanna Burton, Gwendolyn Gerner, Joseph Scafidi, Xiaobu Ye, Frances Northington, and Shenandoah Robinson; supervision: Shenandoah Robinson and Lauren Jantzie; project administration: Shenandoah Robinson and Lauren Jantzie; funding acquisition: Shenandoah Robinson, Frances Northington, and Lauren Jantzie; approval of the final manuscript: Lauren Jantzie, Sankar Muthukumar, Yuma Kitase, Vikram Vasan, Mohammed Fouda, Sarah Hamimi, Christopher Burkhardt, Vera Joanna Burton, Gwendolyn Gerner, Joseph Scafidi, Xiaobu Ye, Frances Northington, and Shenandoah Robinson; correspondence and material requests: Shenandoah Robinson.

All data generated and analyzed during this study are available from the corresponding author on reasonable request.

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